Abstract:

Calibration and normalization methods for a grating-based sensor design
are disclosed. The sensor may be constructed in a manner optimized for
both label-free and luminescence, e.g. fluorescence, amplification
detection in a single device. Such a sensor, based on grating or another
periodical structure with appropriate coating, dramatically increases the
diversity of applications and allows realizing novel concepts that
provide qualitative and quantitative information/data for each location
or capture element in the sensor surface. The invention takes advantage
of these different modes to carry out a quality control (QC) step and a
calibration of each individual location of the sensor. Thus, the assay
data can be flagged according to their quality and local density
variations, batch variations and variations in the printed deposition of
probes or the materials to the surface can be compensated.

Claims:

1. A method for assessing the immobilization quality and/or quantity of
probes or an array of probes immobilized on a biosensor having a periodic
grating structure and a multitude of probe locations on a surface
thereof, wherein the immobilization quality and/or quantity of the
immobilized probes is assessed individually at each probe location in a
spatially resolved manner prior to the binding of an analyte to the
probes,said method comprising the steps of:(1) obtaining two-dimensional
data and/or images from said biosensor by:(A) in an Evanescent Resonance
mode, exciting of bound luminescence labels bound to the probes and
collecting data of the resulting emissions from said biosensor, and(B) in
a label-free mode, obtaining a two dimensional image of the biosensor
surface and peak wavelength value (PWV) data for the portions of the
two-dimensional image which comprise images of probe locations of the
biosensor, the peak wavelength value comprising the peak wavelength of
light reflected from the biosensor due to resonant coupling of light into
the biosensor; and(2) characterizing the immobilization quality and/or
quantity of the probes or array of probes immobilized on the biosensor
surface from the two-dimensional data and/or images.

2. The method of claim 1, wherein the biosensor comprises a multitude of
sample regions, each sample region potentially containing biological
material bound to the biosensor, and wherein the array is formed as a
surface of a periodic grating structure and wherein step (1) of the
method comprises the steps of:(1) obtaining a two-dimensional image of
the biosensor;(2) obtaining peak wavelength value (PWV) data for the
portions of the two-dimensional image which comprise images of sample
regions of the biosensor, the peak wavelength value comprising the peak
wavelength of light reflected from the biosensor due to resonant coupling
of light into the grating structure; and(3) obtaining quantitative
information as to the amount of binding of the biological material to the
sample regions of the array from the peak wavelength value data.

3. A method for assessing the immobilization quality and/or quantity of
probes or an array of probes immobilized on a biosensor having a periodic
grating structure and a multitude of probe locations on a surface
thereof, wherein the immobilization quality and/or quantity of the probes
is assessed individually at each probe location in a spatially resolved
manner prior to the binding of an analyte to the probes,said method
comprising the steps of:(1) measuring peak wavelength value (PWV) data of
the probe locations of the biosensor;(2) obtaining a 2-dimensional image
of the probe locations in a spatially resolved manner (PWV Images)(3)
obtaining quantitative information of the immobilization quality and/or
quantity of probes immobilized on the biosensor from the PWV data.

4. The method of claim 2, wherein the method further comprises the steps
of:applying a labelled sample to the multitude of probe
locations;obtaining evanescent resonance (ER) measurements of the
multitude of probe locations; andnormalizing the ER measurements with the
quantitative information obtained.

5. The method of claim 4, wherein the normalization of the ER measurements
is according to normalizing equation (1).

6. The method of claim 3, wherein the method further comprises the steps
of:applying a labelled sample to the multitude of probe
locations;obtaining evanescent resonance (ER) measurements of the
multitude of probe locations; andnormalizing the ER measurements with the
quantitative information obtained.

7. The method of claim 6, wherein the normalization of the ER measurements
is according to normalizing equation (1).

8. The method of claim 3, wherein the PWV data and images are indicative
for the amount and morphology of potentially immobilized material on the
surface of the biosensor, and wherein the method further comprises the
step of using the PWV data and images to calibrate data and images
obtained after hybridization of a sample to the immobilized material.

9. The method of claim 1, further comprising the step of acquiring label
free PWV data and images and/or ER measurements at one or more stages in
a process of manufacture of the biosensor, including one or more of the
following stages: biosensor surface cleaning, biosensor surface
modification, immobilization of materials onto the biosensor surface,
biosensor wash steps, biosensor drying steps, and hybridization of a
sample on the surface of the biosensor.

10. The method of claim 8, further comprising the step of correcting data
and images obtained after hybridization based on the pre-hybridisation
PWV images/data, thus calibrating/compensating for variations of amount
and morphology of immobilized capture material immobilised on the
biosensor.

11. The method of claim 2, wherein the biosensor further comprises a
substrate and wherein the surface of the biosensor is coated with a layer
of high index of refraction material consisting of material having a
refractive index n2 higher than that of the substrate n1,
wherein the depth of the layer is between 10 and 1000 nm, and the
resulting periodicity is in the range of 100 to 1000 nm, and the
substrate is of planar, cylindrical, conical, spherical, or elliptical
geometry.

12. The method of claim 1, wherein a salt image is further obtained and
analysed, said salt image resulting from the method of spotting the
probes to be immobilised to the support, said method comprising the steps
of spotting a salt containing-solution containing the probes to be
immobilised to the support, optionally drying said salt
containing-solution containing the probes, and obtaining an image of the
locations where the probes should have been immobilised, said image being
obtained prior to any washing step, so that the absence of salt at a
specific location indicates that spotting of the probe to be immobilised
at this specific location has not occurred at said specific location.

14. The method of claim 1, wherein the biosensor includes an optically
transparent layer is formed from inorganic material selected from the
group consisting of a metal oxide such as Ta2O5, TiO2,
Nb2O5, ZrO2, ZnO or HfO2; organic materials selected
from the group consisting of polyamide, polyimide, PP, PS, PMMA,
polyacryl acids, polyacryl esters, polythioether, or
poly(phenylenesulfide); and derivatives thereof.

20. The method of claim 1, further comprising the step of obtaining a
spectrum for background signals produced by the biosensor and wherein the
characterization of the immobilization quality and/or quantity is made
after subtraction of said spectrum for background signals produced by the
biosensor.

21. A method of detecting and/or quantifying an analyte using an biosensor
comprising an array of immobilised probes on a surface thereof, the
biosensor constructed in the form of a periodic grating structure,
wherein the presence and/or concentration of said analyte is normalised
with respect to the presence and/or concentration of said immobilised
probes, wherein said presence and/or concentration of said immobilised
probes is assessed at locations of the array prior to the potential
binding of the analyte to the surface of the biosensor.

22. The method of claim 21, wherein the presence and/or concentration of
the immobilized probes is assessed using a two dimensional image of the
biosensor surface and peak wavelength value (PWV) data for the portions
of the two-dimensional image which comprise images of probe locations of
the biosensor, the peak wavelength value comprising the peak wavelength
of light reflected from the biosensor due to resonant coupling of light
into the biosensor

23. The method of claim 21, further comprising the step of obtaining a
spectrum for background signals produced by the biosensor and wherein the
normalization is performed after subtraction of said spectrum for
background signals produced by the biosensor.

24. The method of claim 21, further comprising the steps of:(1) processing
the biosensor to prepare for a hybridisation of a sample,(2) hybridizing
the sample to the biosensor; and(3) recording a post-hybridisation image
of the biosensor, where the resulting image represents the bound sample.

25. The method of claim 24, further comprising the step of recording a
post-hybridisation label free image in a label-free mode of the
biosensor.

26. The method of claim 24, further comprising performing background
subtraction methods to compensate for background levels of signals
produced by the biosensor.

27. The method of claim 25, further comprising the step of correcting the
post-hybridisation image based on the pre-hybridisation data obtained,
thus compensating for capture material variations on the biosensor.

29. The method of claim 1, wherein the probes are deposited on the
biosensor with a printer.

30. The method of claim 1, wherein the biosensor is attached to an
internal surface of a liquid containing vessel.

31. The method of claim 30, wherein the liquid containing vessel is
selected from the group consisting of a microtitre plate, a test tube, a
Petri dish and a microfluidic channel.

32. A non-contact method of qualitative analysis of a microarray chip,
comprising the steps of:(a) providing a microarray chip in the form of a
multitude of sample regions on a surface of a periodic grating
structure;(b) depositing of capture elements to the grating structure;(c)
obtaining a two-dimensional image of the microarray chip;(d) obtaining
peak wavelength value (PWV) data for the portions of the two-dimensional
image which comprise images of sample regions of the microarray chip, the
peak wavelength value comprising the peak wavelength of light reflected
from the microarray due to resonant coupling of light into the grating
structure; and(e) obtaining qualitative information as to the binding of
the capture elements to the sample regions of the microarray from either
(1) the two-dimensional image or (2) the peak wavelength value data.

33. The method of claim 32, wherein the capture elements are deposited
using a piezo-array printer.

34. The method of claim 32, wherein the material is applied/deposited
using a pin printer.

35. The method of claim 32, wherein the qualitative information obtained
in step e) comprises characterizing the binding of the capture elements
as a function of the position on the surface of the biosensor.

36. The method of claim 32, wherein the capture elements are selected from
the group of materials consisting of a nucleic acid material and a
protein.

37. A method of analysis of a microarray chip comprising the steps of:(a)
providing a microarray chip in the form of a multitude of sample regions
on a surface of a periodic grating structure;(b) applying a biological
material to the sample regions;(c) obtaining a two-dimensional image of
the microarray;(d) obtaining peak wavelength value (PWV) data for the
portions of the two-dimensional image which comprise images of sample
regions of the microarray, the peak wavelength value comprising the peak
wavelength of light reflected from the microarray due to resonant
coupling of light into the grating structure;(e) performing a
hybridisation step comprising applying a second sample material to the
sample regions;(f) obtaining a two-dimensional image of the microarray
after the hybridisation step; and(g) obtaining peak wavelength value
(PWV) data for the portions of the two-dimensional image which comprise
images of sample regions of the microarray after the hybridisation step.

38. The method of claim 37, wherein the hybridisation step comprises the
step of applying a fluorescent probe to the biological material.

39. The method of claim 37, further comprising the step of obtaining
evanescent resonance (ER) measurements of the sample regions after the
hybridisation step.

40. The method of claim 37, further comprising the step of obtaining ER
measurements from the microarray chip and normalizing the measurements
with reference to quantitative data of the amount of biological material
bound to the sample regions obtained from the peak wavelength value (PWV)
data obtained in step d).

41. The method of claim 37, where the biological material adhered to a
biosensor comprises a DNA microarray.

42. A method for the determination of the amount of DNA adhered to a
biosensor following a hybridisation protocol comprising the combined use
of label-free and label methods.

[0002]This invention relates generally to a method for assessing the
quality of an array of probes immobilised on a support, wherein the
presence and/or amount of the immobilised probes is assessed individually
at each location of the array prior to the potential binding of the
labelled analyte.

BACKGROUND OF THE INVENTION

[0003]Microarrays and other assay formats making use of arrays of
materials have become powerful tools to increase data quantity and
quality in various areas, such as the life sciences, pharmaceutical drug
research/development, and recently the clinical environment. Although
considered an important key technology, microarray data still suffer
various experimental error sources that render long term studies and
comparison of data from different laboratories difficult. Production
batch and processing batch variations have to be taken into account
during experimental design to reduce and control experimental variation.

[0004]A key problem for microarray and other array-based technologies is
that the amount of immobilized capture element in certain locations on
the platform might vary over a wide range (including absent or missing),
under the influence of process parameters such as binding capacity of the
surface of the platform, concentration of the used capture element
solutions, temperature, humidity, incubation time, deposition technology,
etc. Quality control of these process steps is therefore very important.

[0005]Following hybridisation, the probe-analyte signal depends on both
the sequence of immobilized oligonucleotide and the quantity of
oligonucleotide immobilized prior to hybridisation. Current methods do
not allow for independent determination of the amount of immobilized
oligonucleotide. Arrays hybridised with labelled random oligonucleotides
specifically designated for calibration do not evenly represent the
various sequences present in the of the array population. Another widely
used method of calibrating/normalising measurements performed with a
microarray or other array based technologies is to make use of reference
samples--mixed with the sample of interest during the hybridisation step,
wherein both samples carry different fluorescence labels (e.g. CY3/green
emission and CY5/red emission). In this method, a constant aliquot of the
reference sample is distributed through the entire experiment as a
reference. This approach does not solve the problem that signal intensity
again, depends on reference sample and capture element sequences. The
amount of probe oligonucleotide cannot be determined. In addition, only
relative calibration is possible and the calibration of
features/sequences of low abundance in the reference sample is poor. For
instance, dye swapping might be required to avoid experimental bias.

[0006]Other approaches, e.g. such as dye labelling/staining technology,
also only assay individual microarrays of a given production batch with
above described disadvantages.

[0007]The methods of this disclosure are suitable for use with
grating-based biosensors which support a label-free detection of a sample
and also luminescence/fluorescence amplification of a sample, referred to
below as Evanescent Resonance (ER) technology. A brief introduction to
both types of sample detection and measurement is set forth below. A
detailed explanation of both technologies and a biosensor structure
designed for both types of detection is set forth in published PCT patent
application WO 2007/019024, the entire contents of which is incorporated
by reference herein.

[0008]Label-Free Detection Sensors

[0009]Grating-based sensors represent a new class of optical devices that
have been enabled by recent advances in semiconductor fabrication tools
with the ability to accurately deposit and etch materials with precision
less than 100 nm.

[0010]Several properties of photonic crystals make them ideal candidates
for application as grating-type label free optical biosensors. First, the
reflectance/transmittance behaviour of a photonic crystal can be readily
manipulated by the adsorption of biological material such as proteins,
DNA, cells, virus particles, and bacteria on the crystal. Other types of
biological entities which can be detected include small and smaller
molecular weight molecules (i.e., substances of molecular weight <1000
Daltons (Da) and between 1000 Da to 10,000 Da), amino acids, nucleic
acids, lipids, carbohydrates, nucleic acid polymers, viral particles,
viral components and cellular components such as but not limited to
vesicles, mitochondria, membranes, structural features, periplasm, or any
extracts thereof. These types of materials have demonstrated the ability
to alter the optical path length of light passing through them by virtue
of their finite dielectric permittivity. Second, the
reflected/transmitted spectra of photonic crystals can be extremely
narrow, enabling high-resolution determination of shifts in their optical
properties due to biochemical binding while using simple illumination and
detection apparatus. Third, photonic crystal structures can be designed
to highly localize electromagnetic field propagation, so that a single
photonic crystal surface can be used to support, in parallel, the
measurement of a large number of biochemical binding events without
optical interference between neighbouring regions within <3-5 microns.
Finally, a wide range of materials and fabrication methods can be
employed to build practical photonic crystal devices with high
surface/volume ratios, and the capability for concentrating the
electromagnetic field intensity in regions in contact with a biochemical
test sample. The materials and fabrication methods can be selected to
optimize high-volume manufacturing using plastic-based materials or
high-sensitivity performance using semiconductor materials.

[0012]The combined advantages of photonic crystal biosensors may not be
exceeded by any other label-free biosensor technique. The development of
highly sensitive, miniature, low cost, highly parallel biosensors and
simple, miniature, and rugged readout instrumentation will enable
biosensors to be applied in the fields of pharmaceutical discovery,
diagnostic testing, environmental testing, and food safety in
applications that have not been economically feasible in the past.

[0013]In order to adapt a photonic bandgap device to perform as a
biosensor, some portion of the structure must be in contact with a test
sample. Biomolecules, cells, proteins, or other substances are introduced
to the portion of the photonic crystal and adsorbed where the locally
confined electromagnetic field intensity is greatest. As a result, the
resonant coupling of light into the crystal is modified, and the
reflected/transmitted output (i.e., peak wavelength) is tuned, i.e.,
shifted. The amount of shift in the reflected output is related to the
amount of substance present on the sensor. The sensors are used in
conjunction with an illumination and detection instrument that directs
light into the sensor and captures the reflected or transmitted light.
The reflected or transmitted light is fed to a spectrometer that measures
the shift in the peak wavelength.

[0014]The ability of photonic crystals to provide high quality factor (Q)
resonant light coupling, high electromagnetic energy density, and tight
optical confinement can also be exploited to produce highly sensitive
biochemical sensors. Here, Q is a measure of the sharpness of the peak
wavelength at the resonant frequency. Photonic crystal biosensors are
designed to allow a test sample to penetrate the periodic lattice, and to
tune the resonant optical coupling condition through modification of the
surface dielectric constant of the crystal through the attachment of
biomolecules or cells. Due to the high Q of the resonance, and the strong
interaction of coupled electromagnetic fields with surface-bound
materials, several of the highest sensitivity biosensor devices reported
are derived from photonic crystals. See the Cunningham et al. papers
cited previously. Such devices have demonstrated the capability for
detecting molecules with molecular weights less than 200 Daltons (Da)
with high signal-to-noise margins, and for detecting individual cells.
Because resonantly-coupled light within a photonic crystal can be
effectively spatially confined, a photonic crystal surface is capable of
supporting large numbers of simultaneous biochemical assays in an array
format, where neighbouring regions within <10 μm of each other can
be measured independently. See Li, P., B. Lin, J. Gerstenmaier, and B. T.
Cunningham, "A new method for label-free imaging of biomolecular
interactions." Sensors and Actuators B, 2003.

[0015]There are many practical benefits for label-free biosensors based on
photonic crystal structures. Direct detection of biochemical and cellular
binding without the use of a fluorophore, radioligand or secondary
reporter removes experimental uncertainty induced by the effect of the
label on molecular conformation, blocking of active binding epitopes,
steric hindrance, inaccessibility of the labelling site, or the inability
to find an appropriate label that functions equivalently for all
molecules in an experiment. Label-free detection methods greatly simplify
the time and effort required for assay development, while removing
experimental artifacts from quenching, shelf life, and background
fluorescence. Compared to other label-free optical biosensors, photonic
crystals are easily queried by simply illuminating at normal incidence
with a broadband light source (such as a light bulb or LED) and measuring
shifts in the reflected colour. The simple excitation/readout scheme
enables low cost, miniature, robust systems that are suitable for use in
laboratory instruments as well as portable handheld systems for
point-of-care medical diagnostics and environmental monitoring. Because
the photonic crystal itself consumes no power, the devices are easily
embedded within a variety of liquid or gas sampling systems, or deployed
in the context of an optical network where a single
illumination/detection base station can track the status of thousands of
sensors within a building. While photonic crystal biosensors can be
fabricated using a wide variety of materials and methods, high
sensitivity structures have been demonstrated using plastic-based
processes that can be performed on continuous sheets of film.
Plastic-based designs and manufacturing methods will enable photonic
crystal biosensors to be used in applications where low cost/assay is
required, that have not been previously economically feasible for other
optical biosensors.

[0016]One of the assignees of the present invention has developed a
photonic crystal biosensor and associated detection instrument for
label-free binding detection (termed BIND). The sensor and detection
instrument are described in the patent literature; see U.S. patent
application publications U.S. 2003/0027327; 2002/0127565, 2003/0059855
and 2003/0032039, and U.S. Pat. No. 7,023,544. Methods for detection of a
shift in the resonant peak wavelength are taught in U.S. Patent
application publication 2003/0077660. The biosensors described in these
references include 1- and 2-dimensional periodic structured surfaces
applied to a continuous sheet of plastic film or substrate. The crystal
resonant wavelength is determined by measuring the peak reflectivity at
normal incidence with a spectrometer to obtain a wavelength resolution of
0.5 picometer. The resulting mass detection sensitivity of <1
pg/mm2 (obtained without 3-dimensional hydrogel surface chemistry)
has not been demonstrated by any other commercially available biosensor.

[0017]A fundamental advantage of the biosensor devices described in the
above-referenced patent applications is the ability to mass-manufacture
with plastic materials in continuous processes at a 1-2 feet/minute rate.
Methods of mass production of the sensors are disclosed in U.S. Patent
application publication 2003/0017581.

[0018]Details on the construction of the system of are set forth in the
published U.S. Patent Application 2003/0059855. Another example of
periodically structures arrays can also be found in WO 01/02839.

[0019]As shown in FIG. 1, the periodic surface structure of a biosensor 10
is fabricated from a low refractive index material 12 that is overcoated
with a thin film of higher refractive index material 14. The low
refractive index material 12 is bonded to a base sheet of clear plastic
material 16. The surface structure is replicated within a layer of cured
epoxy 12 from a silicon-wafer "master" mold (i.e. a negative of the
desired replicated structure) using a continuous-film process on a
polyester substrate 16. The liquid epoxy 12 conforms to the shape of the
master grating, and is subsequently cured by exposure to ultraviolet
light. The cured epoxy 12 preferentially adheres to the sheet 16, and is
peeled away from the silicon wafer. Sensor fabrication was completed by
sputter deposition of 120 nm titanium oxide (TiO2) high index of
refraction material 14 on the cured epoxy 12 grating surface. Following
titanium oxide deposition, 3×5-inch microplate sections are cut
from the sensor sheet, and attached to the bottoms of bottomless 96-well
and 384-well microtitre plates with epoxy.

[0020]As shown in FIG. 2, the wells 20 defining the wells of the
mircotiter plate contain a liquid sample 22. The combination of the
bottomless microplate and the biosensor structure 10 is collectively
shown as biosensor apparatus 26. Using this approach, photonic crystal
sensors are mass produced on a square-yardage basis at very low cost.

[0021]The detection instrument for the photonic crystal biosensor is
simple, inexpensive, low power, and robust. A schematic diagram of the
system is shown in FIG. 2. In order to detect the reflected resonance, a
white light source illuminates a ˜1 mm diameter region of the
sensor surface through a 100 micrometer diameter fiber optic 32 and a
collimating lens 34 at nominally normal incidence through the bottom of
the microplate. A detection fiber 36 is bundled with the illumination
fiber 32 for gathering reflected light for analysis with a spectrometer
38. A series of 8 illumination/detection heads 40 are arranged in a
linear fashion, so that reflection spectra are gathered from all 8 wells
in a microplate column at once. See FIG. 3. The microplate+biosensor 10
sit upon an X-Y addressable motion stage (not shown in FIG. 2) so that
each column of wells in the microplate can be addressed in sequence. The
instrument measures all 96 wells in ˜15 seconds, limited by the
rate of the motion stage. Further details on the construction of the
system of FIGS. 2 and 3 are set forth in the published U.S. Patent
Application 2003/0059855.

[0022]Fluorescence Amplification Sensors

[0023]U.S. Pat. No. 6,707,561 describes a grating-based biosensing
technology that is sometimes referred to in the art as Evanescent
Resonance (ER) technology. This technology also employs a sub-micron
scale grating structure to amplify a luminescence signal (e.g.,
fluorescence, chemiluminescence, electroluminescence, phosphorescence
signal), following a binding event on the grating surface, where one of
the bound molecules carries a fluorescent label. ER technology enhances
the sensitivity of fluorophore based assays enabling binding detection at
analyte concentrations significantly lower than non-amplified assays.

[0024]ER technology makes use of an optical grating in combination with a
high refractive index coating (for details see below) to generate optical
resonance and to concentrate laser light on the sensor surface where
binding has taken place. In practice, a laser scanner scans the sensor at
some angle of incidence (theta), typically from above the grating, while
a detector detects fluoresced light (at longer optical wavelength) from
the sensor surface. Also, non-scanning optical set-ups that use CCD
(Charge Coupled Devices) cameras to measure larger areas of the sensors
at a time can be configured to generate evancesent resonance. By design,
ER sensor optical properties result in nearly 100% reflection, also
attributed as resonance, at a specific angle of incidence and laser
wavelength (λ). Confinement of the laser light by and within the
grating structure amplifies emission from fluorophores bound within range
of the evanescent field (typically 1-2 um). Hence, at resonance,
transmitted light intensity drops to near zero.

[0025]As noted above, the label-free biosensors described in the
above-referenced patent applications employ a sub-micron scale grating
structure but typically with a significantly different grating geometry
and objective as compared to gratings intended for ER use. In practical
use, label-free and ER technologies have different requirements for
optical characteristics near resonance. The spectral width and location
of the resonance phenomena describes the primary difference. Resonance
width refers to the full width at half maximum, in wavelength measure, of
a resonance feature plotted as reflectance (or transmittance) versus
wavelength (also referred to as Q factor above). Resonance width can also
refer to the width, in degrees, of a resonance feature plotted on a curve
representing reflectance or transmittance as a function of theta, where
theta is the angle of incident light.

[0026]Optimally, a label-free grating-based sensor produces as narrow a
resonance peak as possible, to facilitate detection of small changes in
peak position indicating low binding events. A label-free sensor also
benefits from a high grating surface area in order to bind more material.
In current practice, one achieves higher surface area by making the
grating deeper (though other approaches exist). Current commercial
embodiments of label-free sensors produce a resonance near 850 nm, thus
BIND label-free detection instrumentation has been optimized to read this
wavelength.

[0027]Conversely, practical ER grating sensor designs employ a relatively
broad resonance to ensure that resonance occurs at the fixed wavelength
laser light and often fixed angle of incidence in the presence of
physical variables such as material accumulation on the grating or
variation in sensor manufacture. Because field strength generally
decreases with resonance width, practical ER sensor design calls for a
balance in resonance width. By choosing an appropriate ER resonance
width, one ensures consistent amplification across a range of assay,
instrument and sensor variables while maintaining ER signal gain. A
typical application uses a 633 nm wavelength to excite a popular
fluorescent dye, known in the art as Cy5. Some ER scanning
instrumentation permits adjustments to incident angle to "tune" the
resonance towards maximum laser fluorophore coupling. This practice,
however, may induce an unacceptable source of variation without proper
controls.

[0028]Known ER designs also employ more shallow grating depths than
optimal label-free designs. For example, the above-referenced U.S. Pat.
No. 6,707,561 specifies the ratio of grating depth to "transparent layer"
(i.e., high index coating layer) thickness of less than 1 and more
preferably between 0.3 and 0.7. Optimal label-free designs employ
gratings with a similarly defined ratio of greater than 1 and preferably
greater than 1.5. Label-free designs typically define grating depth in
terms of the grating line width or half period. For example, currently
practiced commercial label-free sensors have a half period of 275 nm and
a grating depth of approximately 275 nm, thus describing a 1:1 geometric
ratio. This same sensor design employs a high index of refraction oxide
coating on top of the grating with a thickness of approximately 90 nm.
Thus, according to the definition in the U.S. Pat. No. 6,707,561, this
sensor has a grating depth:oxide thickness ratio of approximately 3:1.

SUMMARY OF THE INVENTION

[0029]The present disclosure provides for calibration and normalization
uses of grating-based biosensor designs. The biosensors may be
constructed in a manner such that that the biosensor is optimized for
both modes of detection (label-free and luminescence, e.g. fluorescence,
amplification), in a single device. Such a sensor, based on grating or
another periodical structure with appropriate coating, dramatically
increases the diversity of applications and allows realizing novel
concepts that provide qualitative and quantitative information/data for
each location in the microarray/biosensor.

[0030]The present disclosure takes advantage from these different modes to
carry out quality control (QC) steps at various stages of the sensor
preparation and a calibration of each individual location of the
biosensor. Thus, the assay data can be flagged according to their quality
and local density variations, and batch or printing variations of the
biosensor can be compensated.

[0031]The present disclosure makes use of the ER and label-free
technologies in a combination that allows obtaining
qualitative/quantitative information as well as calibration for all
capture elements immobilized and for all microarrays that are produced in
a batch (intra batch). The methods of the present invention also allow
compensating for production batch differences (inter batch). The
biosensors for use in the inventive methods may be based on periodically
structured microarrays substrates with thin dielectric coatings, as
described in e.g. patents WO01/02839, US2003/0027327; US2002/0127565,
US2003/0059855 or US2003/0032039, or in U.S. Pat. Nos. 6,707,561 or
7,023,544. These structures can also be described as photonic crystals or
photonic band gap materials.

[0032]In one aspect, the present disclosure provides a method for
assessing the immobilisation quality and/or quantity of the probes of an
array of probes immobilised on a support. The presence and/or amount of
the immobilised probes are assessed individually at each location of the
array in a spatially resolved manner prior to the potential binding of
the analyte.

[0033]In one embodiment, the support has an optically transparent
substrate having a refractive index n1, and a non-metallic optically
transparent layer formed on the surface of the substrate, said layer
having a refractive index n2 which is greater than n1, wherein
said support incorporates therein one or more grating or corrugated
structures which define one or more sensing areas or regions, each for
one or multiple capture elements or locations, wherein said corrugated
structure comprising periodic grooves. The depth of the grooves is in the
range of 3 nm to the thickness of the optically transparent layer. The
thickness of the optically transparent layer is in the range of 30 to
1000 mn. The period of the corrugated structure is in the range of 200 to
1000 nm, the ratio of groove depth to the thickness of the optically
transparent layer is in the range of 0.02 to 1, and the ratio of groove
width to the period of the grooves is in the range of 0.2 to 0.8. Thus,
in an ER mode, coherent light incident on said support at an appropriate
angle can be diffracted into individual beams or diffraction orders which
interfere resulting in reduction of the transmitted beam and an abnormal
high reflection of the incident light, thereby generating an evanescent
field at the surface of the one or multiple sensing areas. Alternatively,
coherent and linearly polarised light incident on the platform at an
appropriate angle can be diffracted into individual beams or diffraction
orders which interfere resulting in almost total extinction of the
transmitted beam and an abnormal high reflection of the incident light,
thereby generating an evanescent field at the surface of the one or
multiple sensing areas or locations.

[0034]The presence and/or amount of each of the materials immobilized on
the support or biosensor surface is measured in a label-free mode,
comprising measurement of peak wavelength value (PWV) data of the support
or regions of the support at all stages of the processes used for the
production/preparation, in particular prior and post material
immobilisation (printing/arraying, microarray production), wherein
changes of the PWV data can be used to construct 2-dimensional images of
the transducer in spatially resolved manner (PWV images). These images
provide quantitative information of the respective process steps. In
particular, the PWV data/images are indicative for the amount and
morphology of potentially immobilized material. The PWV data/images can
be used to quantify the immobilized material on the transducer and can be
used for assessment of quality, spatially resolved quantification of
material immobilized on the biosensor, and can be used in downstream
data/image processing to calibrate data and images obtained after
hybridisation with labelled sample (luminescence, fluorescence or other
labels).

[0035]In one embodiment, the present disclosure provides a method wherein
the label-free PWV data/images and/or ER measurements can be carried out
at all stages of the process, prior/post the support/transducer surface
cleaning, surface modification, immobilisation of materials, wash steps,
drying steps, hybridisation of sample; independent from the
sequence/order, wherein processing steps can also be repeated or used at
several stages of the process in an adapted/suitable way.

[0036]In another embodiment, the present disclosure provides a method
wherein the signals of the post-hybridisation images steps can be
corrected based on the pre-hybridisation PWV images/data, thus
calibrating/compensating for variations of amount and morphology of
immobilized capture material immobilised on the biosensor.

[0037]In yet another embodiment, the present disclosure provides a method
wherein the sensor surface is coated with a layer of nanoparticles
consisting of material having a refractive index n2 higher than that
of the substrate n1. The nanoparticles attached to the surface are
of similar size and act as periodical structure/arrangement that allows
optical coupling/resonance of the device/transducer/support as described
herein. The size of the nanoparticles is preferentially between 10 and
1000 nm, and the resulting periodicity is in the range of 100 to 1000 nm,
and the substrate is of planar, cylindrical, conical, spherical, or
elliptical geometry.

[0038]In still another embodiment, the present disclosure provides a
method wherein a salt image is further obtained of the biosensor and
analysed. The salt image results from a method of spotting the probes to
be immobilised to the support. The method of spotting includes steps of
spotting a salt containing-solution containing the probes to be
immobilised to the support, optionally drying the salt
containing-solution containing the probes, and obtaining an image of the
locations where the probes should have been immobilised. The image is
obtained prior to any washing step, so that the absence of salt at a
specific location indicates that spotting of the probe to be immobilised
at this specific location has not occurred.

[0044]The method can also involve a step of obtaining spectrum/data for
background signals produced by the biosensor and wherein the quantitative
information is obtained after subtraction of the spectrum for background
signals.

[0045]In another aspect, the present disclosure provides a method of
detecting and/or quantifying an analyte using an array of immobilised
probes, wherein the presence and/or concentration of the analyte is
normalised with respect to the presence and/or concentration of said
immobilised probes, wherein the presence and/or concentration of the
immobilised probes is assessed individually at each location of the array
prior to the potential binding of the analyte.

[0046]In one embodiment, the background subtraction methods are applied to
compensate for background levels for all types of images. Any suitable
background subtraction method known in the art may be used, and the
details are not particularly important.

[0047]In another embodiment, the data/images obtained at different stages
of the process can be used to calculate new images or data using suitable
algorithms, e.g. for calibration, and/or background correction. The
microarray may be preprocessed or processed to prepare the hybridization
of a sample. The sample is then hybridized to the microarray/support, a
post-hybridisation process for the microarray/support is applied, and a
luminescence-based post-hybridisation image is recorded in luminescence
mode of the microarray/support. The resulting image represents the bound
luminescence-labelled analyte material. Alternatively or additionally, a
post-hybridisation label free image can be recorded in the label-free
mode. This represents the immobilized material/capture probes and the
bound material originating from the sample incubation.

[0048]The background subtraction methods may be applied to compensate for
background levels for all types of images. In addition, the signals of
the post-hybridisation images can be corrected based on the
pre-hybridisation data obtained previously, thus calibrating/compensating
for capture material variations on the microarray.

[0049]In yet another aspect, the present disclosure provides a method of
analysis of a microarray comprising a multitude of sample regions, each
sample region potentially containing labelled or unlabeled capture probes
bound to the microarray, and wherein the microarray is formed as a
surface of a periodic grating structure. The steps of the method include:
(a) obtaining a two-dimensional image of the microarray; (b) obtaining
peak wavelength value (PWV) data for the portions of the image which
comprise images of capture probe regions of the array, the peak
wavelength value comprising the peak wavelength of light reflected from
the array due to resonant coupling of light into the grating structure;
and (c) obtaining quantitative information as to the amount of
binding/deposition/immobilisation of the capture probe to the sample
regions of the array from the peak wavelength value data.

[0050]In another embodiment, the method further comprises the steps of (d)
applying labelled samples (e.g. luminescence fluorescence) to the sample
regions; (e) obtaining evanescent resonance (ER) measurements of the
sample regions; and (f) normalizing the ER measurements with the
quantitative information obtained in step c).

[0051]In yet another embodiment, the method further includes the step of
obtaining PWV data for background signals produced by the microarray and
wherein the quantitative information in step c) is made after subtraction
of the PWV data for the background signals.

[0052]The labelled samples can be selected from the group of materials
consisting of nucleic acids, proteins and protein fragments, peptides,
any biological relevant binding partners, cells or fragments thereof,
chemical sensing compounds, etc.

[0053]In still another embodiment, the method further includes the step of
obtaining qualitative or quantitative data as to the
binding/immobilisation of sample material to the array from analysis of
either the two-dimensional image or the peak wavelength value data.

[0054]The array elements (capture elements) can be applied to the
microarray/biosensor/substrate platform with a suitable device or
systems, such as for example a microarray printer, a Pin Printer, an
Ink-Jet printer, a photoimmobilisation system, or other technique either
known in the art or later developed. In one embodiment, the capture
elements material is applied or deposited using a piezo-array printer.

[0055]In another aspect, the present disclosure provides a non-contact
method of qualitative analysis of a printed microarray chip, including
the steps of (a) providing a microarray or biosensor in the form of a
multitude of sample regions on a surface of a periodic grating structure;
(b) deposition of capture elements to the microarray; (c) obtaining a
two-dimensional image of the microarray; (d) obtaining peak wavelength
value (PWV) data for the portions of the two-dimensional image which
comprise images of sample regions of the microarray, the peak wavelength
value comprising the peak wavelength of light reflected from the
microarray due to resonant coupling of light into the grating structure;
and (e) obtaining qualitative information as to the binding of the
capture elements to/on the sample regions of the microarray from either
(1) the two-dimensional image or (2) the peak wavelength value data.

[0056]The qualitative or quantitative information obtained by the method
in step (e) can be by determining the amount of bound/immobilized
material as a function of the position on the substrate.

[0057]The capture elements can be selected from the group of materials
consisting of a nucleic acid material and a protein or
chemical/biological/physically/optically modified derivatives/fragments
thereof. In general, any capture element of organic, inorganic,
biological or chemical nature can be used for sensor preparation.

[0058]In yet another aspect, the present disclosure provides a method of
analysis of a microarray chip including the steps of: (a) providing a
microarray chip in the form of a multitude of immobilized capture
elements on a surface of a periodic grating structure; (b) applying a
biological material/sample to the immobilization capture elements; (c)
obtaining a two-dimensional image of the microarray; (d) obtaining peak
wavelength value (PWV) data for the portions of the two-dimensional image
which comprise images of sample regions of the microarray, the peak
wavelength value comprising the peak wavelength of light reflected from
the microarray due to resonant coupling of light into the grating
structure; (e) performing a hybridisation step comprising applying a
sample material to the immobilization capture elements; (f) obtaining a
two-dimensional image of the microarray after the hybridisation step; and
(g) obtaining peak wavelength value (PWV) data for the portions of the
two-dimensional image which comprise images of sample regions of the
microarray after the hybridisation step.

[0059]The hybridisation step can include applying a luminescent or
fluorescent probe to the biological material. Obtaining evanescent
resonance (ER) measurements of the sample regions can be done after the
hybridisation step. The ER measurements are normalized with reference to
quantitative data of the amount of biological material bound to the
sample regions obtained from the peak wavelength value (PWV) data
obtained in step (d).

[0060]The present disclosure provides a method for the quality control of
a DNA microarray where statistical robustness criteria are established
for the use of the quality control data determined by label-free methods.

[0061]The present disclosure further provides a method for the
determination of the amount of DNA adhered to a biosensor prior to and
following various protocols/processes (e.g., hybridisation) comprising
the use of label-free and label methods.

DESCRIPTION OF THE FIGURES

[0062]Exemplary embodiments are illustrated in the drawings. It is
intended that the embodiments and figures disclosed herein are to be
considered illustrative rather than restrictive.

[0063]FIG. 1 is an illustration of a prior art grating-based biosensor
arrangement.

[0064]FIG. 2 is an illustration of a prior art biosensor and detection
system for illuminating the biosensor and measuring shifts in the peak
wavelength of reflected light from the biosensor.

[0065]FIG. 3 is an illustration of an arrangement of 8 illumination heads
that read an entire row of wells of a biosensor device comprising the
structure of FIG. 1 affixed to the bottom of bottomless microtitre plate
or other suitable liquid-containing vessel.

[0066]FIGS. 4A and 4B are perspective and cross-sectional views,
respectively, of a two-dimensional grating design characterized by
periodic holes in a grating structure which is optimized for BIND
(label-free) detection in a water environment when illuminated by X
polarized light and optimized for ER detection in an air environment when
illuminated by Y polarized light.

[0067]FIGS. 5A and 5B are perspective and cross-sectional views,
respectively, of a two-dimensional grating design characterized by
periodic posts in a grating structure which is optimized in one direction
for BIND (label-free) detection in a water environment when illuminated
by X polarized light and optimized for ER detection in an air environment
when illuminated by Y polarized light.

[0068]FIGS. 6A-6C are three views of a unit cell showing a two-level,
two-dimensional grating structure for yet another embodiment of a
combined ER and label-free sensor.

[0069]FIG. 7 is a schematic drawing of an imaging readout system for a
combined ER and label-free grating-based sensor.

[0070]FIG. 8 is an image of a biosensor in the form of a microarray which
is captured by the CCD camera in a detection system (such as shown in
FIG. 7), the biosensor having a periodic grating structure such as shown
in FIG. 11, having a multiplicity of capture elements.

[0071]FIGS. 9A-9C shows three different images of a portion of one of the
capture element regions of the biosensor of FIG. 8, in which individual
capture element locations are indicated by a circle in FIGS. 9A, 9B, and
9C.

[0072]FIG. 9A is a "salt image" of one of the regions of FIG. 8 which
shows the printed spots or locations that are expected to consist of
capture elements (e.g. oligonucleotide) and print buffer. The actual
morphology of the spot locations in the image is varying and depends on
the process conditions. The image provides only qualitative information,
e.g. spots where capture materials have not been printed/deposited
correctly on the chip surface. This information can be used as basic
quality flags for each of the capture elements.

[0073]FIG. 9b is a label-free, PWV image of the region of FIG. 9A,
obtained after a washing step that removes the excess material from the
biosensor surface. The intensity of the individual locations corresponds
to the amount of immobilized material of the oligonucleotides which are
immobilized/deposited in the individual locations on the microarray. The
intensity measurements ("BIND" data) are made using the system of either
FIG. 3 or 7, or in another type of detection system. The shift in peak
resonance wavelength measured by the detection system is determined on a
pixel-by-pixel basis and the magnitude of such shifts is converted either
to colours or to relative brightness, or both, for purposes of rendering
the label-free image of FIG. 9B.

[0074]FIG. 9C is an image obtained with the system of FIG. 7 in an ER mode
showing the fluorescence intensity of the regions corresponds to the
abundance of a particular mRNA in the used sample.

[0075]FIG. 10 illustrates alternative forms of a biosensor platform,
including: discs, concentric designs, etc., as shown in International
Patent Application WO 01/02839. The sensing elements (see FIG. 10a) can
be arranged in various ways, for instance rectangular, circular,
hexagonal-centric, ellipsoidal, linear or labyrinthine. The sensing area
(see FIG. 10b) may be rectangular, round or of any other shape. The
grooves may be arranged either equidistant linear or equidistant
circular, or may correspond to segments of such structures. The platform
(see FIGS. 10c to 10f) can be either rectangular or disc-shaped, or of
any other geometry. The platform can comprise one or multiple sensing
areas, each sensing area can comprise one or multiple capture elements,
and each capture element can comprise one or multiple labeled or
unlabelled capture molecules. The platform can also be adapted to
microtitre-type plates/devices (see FIG. 10g) in order to perform one or
multiple assays in the individual microtitre wells. This can be achieved
for all plate types: 96, 384, 1536, or higher numbers of wells,
independently of the dimensions of the respective microtitre-plate.

[0076]FIG. 11 shows a schematic view of an ER sensor similar to that shown
in FIGS. 8 and 9.

[0077]FIG. 12 shows a schematic view of an ER/BIND composite sensor.

[0078]FIGS. 13A and B are two views of a unit cell of an ER/BIND composite
sensor as shown in FIG. 12, where the grating depth<thickness of high
refractive index layer.

[0079]FIGS. 14A and 14B are two views of a unit cell of an ER/BIND
composite sensor as shown in FIG. 12, where the grating
depth>thickness of high refractive index layer.

DETAILED DESCRIPTION

[0080]The methods of this disclosure may use a biosensor constructed as a
periodic surface grating in which so-called evanescent resonance can be
created. Evanescent resonance is a phenomenon which has been described
theoretically in the prior art for example in a paper entitled "Theory
and applications of guided mode resonance filters" by S S Wang & R
Magnusson in Applied Optics, 32(14): 2606 to 2613 (10 May 1993) and in a
paper entitled "Coupling gratings as waveguide functional elements" by O.
Parriaux et al, Pure & Applied Optics 5: 453-469 (1996). As explained in
these papers resonance phenomena can occur in planar dielectric layer
diffraction gratings where almost 100% switching of optical energy
between reflected and transmitted waves occurs when the grooves of the
diffraction grating have sufficient depth and the radiation incident on
the corrugated structure is at a particular angle. This phenomenon is
exploited in the sensing area of the platform where that sensing area
includes diffraction grooves of sufficient depth and light is caused to
be incident on the sensing area of the platform at an angle such that
evanescent resonance occurs in that sensing region. This creates in the
sensing region an enhanced evanescent field which is used to excite
samples under investigation. It should be noted that the 100% switching
referred to above occur with parallel beam and linearly polarised
coherent light and the effect of an enhanced evanescent field can also be
achieved with non-polarised light of a non-parallel focussed laser beam.
Excitation photons incident on the chip under resonance conditions couple
onto a thin corrugated metal oxide surface at the site of incidence. As a
result of the transducer geometry, the energy is locally confined into
the thin corrugated layer of high refractive index material.
Consequently, strong electromagnetic fields are generated at the surface
of the chip. The effect has been attributed as evanescent resonance and
leads to increased fluorescence intensity of chromophores close to the
surface. The effective field strength can be increased up to 100-fold by
the confinement of the available excitation energy, depending on the
optical properties of the used optical detection system.

[0081]At resonance conditions, the individual beams interfere in such a
way that the transmitted beam is cancelled out (destructive interference)
and the reflected beams interfere constructively, giving rise to abnormal
high reflection. By choosing appropriate parameters for the above
mentioned corrugated layer structure the excitation energy remains highly
localized. Such structures are described in the literature as photonic
band gap structures, materials with periodic spatial variations of their
refractive index such that electromagnetic radiation cannot propagate in
a particular direction for a particular range of wavelengths. Photonic
bandgap structures allow highly localized modes to appear, see e.g. the
paper entitled "Localisation of One Photon States" by C. Adlard, E. R.
Pike & S. Sarkar in Physical Review Letters, Vol. 79, No 9, pages 1585-87
(1997). Such structures exhibit extremely large propagation losses
corresponding to a mode localisation. The biosensor or transducer (both
terms are used interchangeably herein) of the present disclosure can be
considered as optically active in contrast to optically passive platforms
constructed from e.g. a glass or polymer. Here, optically active means
increasing the electromagnetic field of the excitation beam by energy
confinement.

[0082]The substrate of the biosensor may be formed from inorganic
materials such as glass, SiO2, quartz, silicon, and of different organic
and inorganic components or layer as composite materials. Alternatively
the substrate can be formed from organic materials such as polymers
preferably polycarbonate (PC), poly (methyl methacrylate) (PMMA),
polyimide (PI), polystyrene (PS), polyethylene (PE), polyethylene
terephthalate (PET) or polyurethane (PU). Substrate materials can also
include polycarbonate or cyclo-olefin polymers such as Zeanor®.

[0083]These organic materials are especially preferred for point-of-care
(POC) and personalized medical applications since glass is not accepted
in such an environment. Plastics substrates can be structured (e.g.
embossed) much more easily than glass.

[0084]The non-metallic optically transparent layer may be formed from
inorganic material. Alternatively it can be formed from organic material.
In one example the optically transparent layer is a metal oxide such as
Ta2O5, TiO2, Nb2O5, ZrO2, ZnO or HfO2.

[0086]The depth of the periodic grating or grooves is in the range 3 nm to
the thickness of the optically transparent layer and preferably 10 nm to
the thickness of the optically transparent layer, e.g. 30 nm to the
thickness of the optically transparent layer. The thickness of the
optically transparent layer is in the range 30 to 1000 nm, e.g. 50 to 300
nm, preferably 50-200 nm, the period of the corrugated structure may be
in the range 200 to 1000 nm, e.g. 200 to 500 mm, preferably 250-500 nm,
the ratio of the groove depth to the thickness of the optically
transparent layer lies in the range 0.02 to 1 e.g. 0.25 to 1, preferably
0.3 to 0.7, and the ratio of the grooves width to the period of the
grooves ("duty-cycle") lies in the range 0.2 to 0.8, e.g. 0.4 to 0.6.

[0087]The grooves may be generally rectangular in cross-section.
Alternatively, the grooves may be sinusoidal or of saw tooth
cross-section. The surface structure may be generally symmetrical.
Preferred geometries include rectangular, sinusoidal and trapezoidal
cross-sections. Alternatively, the grooves may be of saw tooth
cross-section (blazed grating) or of other asymmetrical geometry. In
another aspect the groove depth may vary, e.g. in periodic modulations.

[0088]The support or platform may be square or rectangular and the grooves
may extend linearly along the platform so as to cover the surface.
Alternatively the platform may be disc shaped and the grooves may be
circular or linear.

[0089]The grooves (or raised portions) may be formed on a surface of the
substrate. Alternatively the grooves may be formed on a surface of the
optically transparent layer. As a further alternative, grooves may be
formed both on the surface of the substrate which is the interface and on
the surface of the optically transparent layer. The grating structure can
take variety of one and two dimensional forms, including two-level, two
dimensional gratings, as disclosed in published PCT application WO
2002/0179024, the contents of which are incorporated by reference herein.

[0090]The corrugated surface of a single sensing area may be optimized for
one particular excitation wavelength and for one particular type of
polarisation. By appropriate means, e.g. superposition of several
periodic structures which are parallel or perpendicular one with another,
periodic surface relief can be obtained that are suitable for multiple
wavelength use of the platform ("multicolour" applications).
Alternatively, individual sensing areas on one platform may be optimized
for different wavelengths and/or polarisation orientations.

[0091]The design of corrugated (grating) surface can be developed and its
performance modelled with the aid of a computer and a software program
GSolver (Grating Solver Development Co., Allen Tex., www.gsolver.com).
The various geometrical dimensions and parameters, spacing, well depth,
materials, and index of refraction data associated with the materials
allows the design to be studied on a computer and simulations run to
predict the Transmission v. Theta curve and reflection as a function of
wavelength curve. Such simulations can be run in situations where the
sample is dry and when the sample is suspended in water or other fluid
medium with known index of refraction. Such simulations allow the
designer to optimize (i.e., change) the various design parameters
(thicknesses, transitions, period, etc.) to satisfy the requirements for
both ER and label-free detection.

[0092]The present inventive calibration and normalization methods can be
performed using a biosensor constructed in a manner which is optimized
for both label free (BIND) and ER measurements. Such a biosensor can have
a one-dimensional grating structure in the horizontal plane, a two
dimensional grating structure, or two-level, two dimensional grating
structures. Several possible and nonlimiting examples of such a biosensor
will be described in conjunction with FIGS. 4A-4B, 4A-5B and 6A-6C.

[0093]FIGS. 4A and 4B show one specific example of a 2D "holes" embodiment
of a combined BIND and ER biosensor. The biosensor is constructed in two
dimensions so as to be optimized for both ER and label-free (BIND)
detection using a single device. FIGS. 4A and 4B provide perspective and
cross-sectional views, respectively, of a unit cell for a two-dimensional
grating design characterized by periodic holes 210 in a grating
structure. The grating design optimizes for water mode BIND (label-free)
detection and air mode ER detection. The device includes an upper
TiO2 layer 104 of 78 nm thickness and a lower substrate 102 layer of
UV-cured material having a grating pattern as shown applied to a base
substrate sheet.

[0094]The two-dimensional unit cell shown in FIGS. 4A and 4B is designed
in such a way that incident light polarized perpendicular to the X-axis,
as shown, produces a BIND signal, incident light polarized perpendicular
to the Y-axis enables ER measurement. Using this design method, the BIND
and ER resonant wavelengths (at a particular angle of
incidence--preferably near normal incidence) may be chosen independently,
and so the respective BIND and ER resonant wavelengths may occur at very
different values. The combined BIND/ER structure described in this
embodiment is optimized to provide a BIND resonance in the near infrared
(˜800-900 nm) wavelength region, while providing an ER resonance at
632.5 nm for excitation of the Cy5 fluorophore. In this example, the
design assumes a water environment over the sensor during BIND
measurement and an air environment over the sensor during ER measurement.
The differing wavelength requirements for ER and BIND engender selection
of a unit cell with a rectangular "hole" (210). Thus, the unit cell may
have differing dimensions in the X and Y directions. For example, the
period in the X direction is 550 nm for the BIND wavelength, but is 432
nm in the Y direction as required for the lower wavelength ER resonance.
The fabrication process dictates that the high refractive index
dielectric thickness will be the same in the X and Y directions. For
fabrication simplicity, the design also has uniform grating depth. The
fabrication process will also result in rounding of the hole corners,
however the principal function of the design remains unchanged. One
skilled in the art will appreciate that when a computer is used to
generate and test a design such as shown in FIGS. 4A and 4B, the designer
can change the specific dimensions of the unit cell, grating depth, and
coating layers and run simulations of field intensity, peak wavelength,
reflectance as a function of theta, and other tests and may select other
dimensions while still achieving acceptable results. Thus, the example of
FIGS. 4A and 4B is meant to be an illustrative embodiment and not
limiting in scope.

[0095]A 2-dimensional grating structure using a repeating unit cell
characterized by a post will now be described with reference to FIGS.
5A-5B. FIGS. 5A and 5B are perspective and cross-sectional views,
respectively, of a unit cell of 2-dimensional grating design
characterized by periodic posts 220 formed in the sensor surface. Each
unit cell has one post 220. The posts 220 are raised projections in a
substrate material 102 (e.g., UV cured polymer) which is applied to a
base sheet (not shown). A high index of refraction (e.g., TiO2)
coating is applied to the projections and substrate as shown in the
Figures. The structure is optimized for BIND (label-free) detection in a
water environment using light polarized in the X direction and optimized
for ER detection in an air mode, using light polarized in the Y
direction.

[0096]The design of FIGS. 5A and 5B was studied by Rigorous Coupled Wave
Analysis (RCWA) computer simulation. While the previous structure unit
cell of FIGS. 4A and 4B contained a "hole" region surrounded by regions
at a higher plane in the z-direction, the grating structure of FIGS. 5A
and 5B contains a central "post" region, surrounded by regions at a lower
plane in the z-direction. As before, the design of FIGS. 5A and 5B
represents a BIND/ER combined structure that is optimized to provide a
BIND resonance in the near infrared (˜800-900 nm) wavelength
region, while providing an ER at 632 nm for excitation of the Cy5
fluorophore. In this example, the design again assumes a water
environment over the sensor during BIND measurement and an air
environment over the sensor during ER measurement. These differing
wavelength requirements for ER and BIND engender selection of a
rectangular "post" unit cell. Thus, the unit cell may have differing
dimensions in the X and Y directions. For example, the period in the X
direction is 530 nm for the BIND wavelength, but is 414 nm in the Y
direction as required for the lower wavelength ER resonance. The
fabrication process again dictates that the high refractive index
dielectric thickness will be the same in the X and Y directions. For
fabrication simplicity, the design also has uniform grating depth. The
fabrication process will also result in rounding of the post corners,
however the principal function of the design remains unchanged. The
example of FIGS. 5A and 5B is meant as an illustrative example not
limiting in scope. The specific dimensions can of course vary.

[0097]FIGS. 6A-6C are three perspective views of yet another embodiment of
a unit cell 500 for a biosensor grating structure constructed and
designed for a combined ER and label-free (BIND) detection. In order to
appreciate some of the features of this structure, it will be useful to
recapitulate on the design aspects pertinent to evanescent resonance (ER)
and label-free (BIND) sensors. Such sensors differ in three basic design
aspects, namely: resonance wavelength, resonance width, and grating
depth.

[0098]Resonance Wavelength

[0099]The ER sensor prefers resonance to occur in within a few
(˜+/-2) nm of the excitation wavelength. Given that the excitation
light generally comes from a laser and has very narrow bandwidth, this
requirement places high specificity on the wavelength location of the ER
resonance. The BIND mode of operation does not have this limitation and
may benefit from a resonance at another wavelength e.g. outside ambient
lighting wavelength range or to separate the BIND signal spectrally from
the ER excitation source thereby eliminating potential overlapping
detection conflicts.

[0100]Resonance Width

[0101]The ER sensor must have a resonance wide enough for it to overlap
the excitation wavelength in the presence of variables such as biological
coating thickness and illumination numerical aperture. In practice, the
ER resonance should not have a full width at half maximum (FWHM) less
than about 5 nm, and more preferably between 10 and 15 nm. On the other
hand, BIND sensitivity increases approximately as 1/sqrt (FWHM) because
peak location uncertainty decreases as the peak width narrows.

[0102]Grating Depth

[0103]BIND sensors give greater resonance wavelength shift when more
biological material adheres to the grating. A deeper grating offers more
surface area for binding biological material. The ER effect does not
necessarily improve and may degrade as the ER grating depth increases.

[0104]The 2-D designs described previously have uniform grating depth
(e.g. in the post examples the height of the posts, or in the holes
example the depth of the holes). Selecting a single grating depth may
involve a compromise between BIND and ER performance both in terms of
peak width and surface area, i.e. BIND PWV shift.

[0105]The design of the biosensor of FIG. 6A-6C is a two-level,
two-dimensional design. The specifics of the design will be discussed
below in greater detail. This design maintains a narrow TM BIND resonance
and high BIND shift performance, while simultaneous providing a wider TE
ER resonance. Similar to previously described two-dimensional designs,
the BIND and ER gratings can have different periods and hence
independently determined resonance wavelengths.

[0106]This "two level" "comBIND" design of FIG. 6A-6C comprises a
multitude of repeating unit cells 500, each of which superimposes a
relatively shallow ER grating 502 extending in the X direction on a
relatively deep BIND grating 504, extending in the Y direction. FIGS.
6A-6C depict one "unit cell" 500 for this design, which, when replicated
in the XY plane forms the complete grating.

[0107]The unit cell 500 consists of a UV-cured polymer layer 524 which is
applied using a master grating wafer to a base substrate sheet such as
PET film (not shown). The polymer layer 524 has the structure of the BIND
grating 504, namely alternating low and high regions extending in the Y
direction. In the X direction, the grating also has alternating low and
high regions, although the relative height of the high region compared to
the low regions of the UV-cured polymer layer 524 in the X direction is
much less than in the Y direction.

[0108]A TiO2 (or alternatively SiO2 or Ta2O5) layer
522 is deposited over the UV-cured polymer layer. This layer has uniform
thickness in the illustrated embodiment. The layer 522 includes upper
repeating surface 506, 508, 510, and 512, and lower repeating surface
514, 516, 518 and 519. The lower surfaces 514, 516, 518 and 519 are
positioned over the top surface of the UV-cured polymer layer. An air or
water sample medium 520 is placed in contact with the upper surfaces 506,
508, 510, 512 of the TiO2 or SiO2 layer 522.

[0109]As will be appreciated from inspection of FIGS. 6A-6C, the
"two-layer 2-D" grating structure includes a relatively deep BIND grating
504 in the Y dimension, characterized by upper and lower grating surfaces
506/508 and 510/512, respectively. The BIND aspect of the unit cell thus
permits adding or more sample material and allows more material to adhere
to the grating, permitting a greater resonance shift. The deeper grating
in the BIND (Y direction) offers more surface area for binding biological
material.

[0110]The ER grating 502 extending in the X direction, conversely,
consists of a relatively shallow grating pattern with high regions 506
and low regions 508 (and also high region 510 and low region 512). In
addition to providing good BIND detection capability, the grating is
expected to simultaneously provide a wider TE ER resonance with optimal
width.

[0111]Independent operation can also be achieved by using different
excitation angles for laser (ER) and white light source (BIND). The laser
beam or white light source may be directed so that it is incident on the
platform at an angle θ. The angle θ may be defined by the
expression sin θ=n-λΛ where Λ is a period of
the diffractive grooves, λ is the wavelength of the incident light
and n is the effective refractive index of the optically transparent
layer. See, WO 01/02839.

[0112]An apparent advantage of the design of FIGS. 6A-6C is that the ER
and BIND structures should operate independently. Hence, structural
dimensions optimized for either ER detection or BIND detection alone
should work for the combination of the ER and BIND sensor of FIG. 6A-6C.
While the specific dimensions for a structure having the unit cell of
FIG. 6A-6C is of course variable, in one representative embodiment the
BIND grating 504 has a period of between about 260 and about 1500 nm, and
the depth of the grating (distance between surfaces 506 and 510) is
between 100 nm and about 3000 nm. For the ER grating 502, the period is
between about 200 nm and about 1000 nm, and the depth (Z distance between
surfaces 506 and 508, and 510 and 512) is between 10 nm and about 300 nm.

[0113]The structure of FIG. 6A-6C was simulated on a computer using
Rigorous Coupled Wave Analysis (RCWA) and its simulated reflection
spectrum obtained, both with and without the addition of an ER grating
structure in the X direction. Results of the analysis are disclosed in
published PCT application WO 2007/019024 and therefore will not be
discussed here for the sake of brevity.

[0114]ER technology heretofore employs a resonance mode induced by
incident light with a polarisation parallel to the grating, defined here
as TE mode or polarisation. Label-free detection technology typically
employs a resonance mode induced by incident light with polarisation
perpendicular to the grating, defined here as the TM mode or
polarisation. This mode produces the narrowest resonance when the sample
is suspended in a liquid medium. For a 2D grating, this distinction
becomes blurry, and a single peak can be used for both. Also, in theory,
the TM peak could be used for ER and the TE peak could be used for BIND.
TE mode for ER and TM mode for BIND is one possible embodiment and is not
limiting in any way. Above combinations/configurations have the
advantages as described, however, other configurations are also in scope
of this invention.

[0115]During label-free mode detection, biological molecules adhere to the
e.g. TiO2 coating and effectively increase the optical thickness of
that material. This results in a shift in the peak wavelength value (PWV)
of the resonance. A larger PWV shift for a fixed amount of material
represents higher detection sensitivity. When comparing grating designs
in a computer simulation, the simulation of additional biological
material can be modelled by incrementing the thickness of the e.g.
TiO2 layer rather than adding a hypothetical biological layer. This
method has proven effective in other grating design exercises.

[0116]The surface of the optically transparent layer (biosensor substrate)
may include one or a plurality of corrugated sensing areas which each may
carry one or a plurality of capture elements.

[0117]A support used in the method of the invention can further comprise a
cover layer on the surface of a periodic grating opposite of a substrate
layer. Where a cover layer is present, the one or more specific capture
elements are immobilized on the surface of the cover layer opposite of
the two-dimensional grating. Preferably, a cover layer comprises a
material that has a lower refractive index than a material that comprises
the two-dimensional grating, so that the ER performance will be minimally
reduced. A cover layer can be comprised of, for example, glass (including
spin-on glass (SOG)), epoxy, or plastic.

[0118]For example, various polymers that meet the refractive index
requirement of a biosensor can be used for a cover layer. SOG can be used
due to its favourable refractive index, ease of handling, and readiness
of being activated with specific capture elements using the wealth of
glass surface activation techniques. When the flatness of the biosensor
surface is not an issue for a particular system setup, a grating
structure of SiN/glass can directly be used as the sensing surface, the
activation of which can be done using the same means as on a glass
surface.

[0119]Resonant reflection can also be obtained without a planarizing cover
layer over a two-dimensional grating. Hence, a support can contain only a
substrate coated with a structured thin film layer of high refractive
index material. Without the use of a planarizing cover layer, the
surrounding medium (such as air or water) fills the grating. Therefore,
specific capture elements are immobilized to the biosensor on all
surfaces a one or two-dimensional grating exposed to the specific capture
elements, rather than only on an upper surface.

[0120]Each probe/capture element (the terms probes and capture elements
are used interchangeably herein) may contain individual and/or mixtures
of capture molecules which are capable of affinity reactions. The shape
of an individual capture element may be rectangular, circular,
ellipsoidal, or any other shape. The area of an individual capture
element is between 1 μm2 and 10 mm2 e.g. between 20
μm2 and 1 mm2 and preferably between 100 μm2 and 1
mm2. The capture elements may be arranged in a regular two
dimensional array.

[0122]The number of capture elements per sensing region is between 1 and
1,000,000, preferably 1 and 100,000. In another aspect, the number of
capture elements to be immobilized on the platform may not be limited and
may correspond to e.g. the number of genes, DNA sequences, DNA motifs,
DNA micro satellites, single nucleotide polymorphisms (SNPs), proteins or
cell fragments constituting a genome of a species or organism of
interest, or a selection or combination thereof. In a further aspect, the
platform of this invention may contain the genomes of two or more
species, e.g. mouse and rat.

[0124]Preferably, one or more specific capture elements are arranged in a
microarray of distinct locations on a biosensor or transducer. A
microarray of specific capture elements comprises one or more specific
capture elements on a surface of a biosensor of the invention such that a
surface contains many distinct locations, each with a different specific
capture element or with a different amount of a specific capture element.

[0125]For example, an array can comprise 1, 10, 100, 1,000, 10,000,
100,000, or 1,000,000 distinct locations. Such a biosensor surface is
called a microarray because one or more specific capture elements are
typically laid out in a regular grid pattern in x-y coordinates. However,
a microarray to be used in the method of the invention can comprise one
or more specific binding substance laid out in any type of regular or
irregular pattern. For example, distinct locations can define a
microarray of spots of one or more specific capture elements. A
microarray spot can be about 20 to about 500 μm in diameter. A
microarray spot can also be about 50 to about 200 μm in diameter. One
or more specific capture elements can be bound to their specific binding
partners.

[0126]A microarray on a support to be used in a method of the invention
can be created by placing microdroplets of one or more specific capture
elements onto, for example, an x-y grid of locations on a two-dimensional
grating or cover layer surface. When the biosensor is exposed to a test
sample comprising one or more binding partners, the binding partners will
be preferentially attracted to distinct locations on the microarray that
comprise specific capture elements that have high affinity for the
binding partners. Some of the distinct locations will gather binding
partners onto their surface, while other locations will not.

[0127]A specific capture element specifically binds to a binding partner
that is added to the surface of a support to be used in a method of the
invention. A specific capture element specifically binds to its binding
partner, but does not substantially bind other binding partners added to
the surface of a biosensor. For example, where the specific capture
element is an antibody and its binding partner is a particular antigen,
the antibody specifically binds to the particular antigen, but does not
substantially bind other antigens. A binding partner can be, for example,
a nucleic acid, polypeptide, antigen, polyclonal antibody, monoclonal
antibody, single chain antibody (scFv), F(ab) fragment, F(ab') 2
fragment, Fv fragment, small organic molecule, cell, virus, bacteria, and
biological sample. A biological sample can be, for example, blood,
plasma, serum, gastrointestinal secretions, homogenates of tissues or
tumours, synovial fluid, faces, saliva, sputum, cyst fluid, amniotic
fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen,
lymphatic fluid, tears, and prostatic fluid.

[0128]One example of a microarray to be used in a method according to the
present invention is a nucleic acid microarray, in which each distinct
location within the array contains a different nucleic acid molecule. In
this embodiment, the spots within the nucleic acid microarray detect
complementary chemical binding with an opposing strand of a nucleic acid
in a test sample.

[0129]The support/platform may include an adhesion promoting layer
disposed at the surface of the optically transparent layer in order to
enable immobilisation of capture molecules. The adhesion promoting layer
may also comprise a microporous layer (ceramics, glass, Si) for further
increasing assay and detection efficacy or of gel layers which either can
be used as medium for carrying out the capture element immobilisation and
sample analysis, thereby further increasing the assay and detection
efficacy, or which allow separation of analyte mixtures in the sense of
gel electrophoresis. The platform may be formed with a plurality of
sensing areas or regions, each having its own diffractive grooves.

[0130]In other words, immobilisation of one or more capture
elements/probes onto a support/biosensor is performed so that a specific
capture element will not be washed away by rinsing procedures, and so
that its binding to binding partners in a test sample is unimpeded by the
biosensor surface. Several different types of surface chemistry
strategies have been implemented for covalent attachment of specific
capture elements to, for example, glass for use in various types of
microarrays and biosensors. These same methods can be readily adapted to
a biosensor of the invention. Surface preparation of a biosensor so that
it contains the correct functional groups for binding one or more
specific capture element is an integral part of the biosensor
manufacturing process.

[0131]One or more specific capture elements can hence be attached to a
biosensor surface by physical adsorption (i.e., without the use of
chemical linkers), by chemical binding (i.e., with the use of chemical
linkers) or by electrostatic/coulombic interaction. Chemical binding can
generate stronger attachment of specific capture elements on a biosensor
surface and provide defined orientation and conformation of the
surface-bound molecules. For instance, some types of chemical binding
include, for example, amine activation, aldehyde activation, and nickel
activation. These surfaces can be used to attach several different types
of chemical linkers to a biosensor surface. While an amine surface can be
used to attach several types of linker molecules, an aldehyde surface can
be used to bind proteins directly, without an additional linker. A nickel
surface can be used to bind molecules that have an incorporated histidine
("his") tag. Detection of "his-tagged" molecules with a nickel-activated
surface is well known in the art (Whitesides, Anal. Chem. 68, 490
(1996)).

[0132]Immobilisation of specific capture elements to plastic, epoxy, or
high refractive index material can be performed essentially as described
for immobilisation to glass. However, the acid wash step can be
eliminated where such a treatment would damage the material to which the
specific capture elements are immobilized.

[0133]For the detection of binding partners (analytes in label-free mode)
at concentrations less than about 0.1 ng/ml, it is possible to amplify
and transduce binding partners bound to a biosensor into an additional
layer on the biosensor surface. The increased mass deposited on the
biosensor can be easily detected as a consequence of increased optical
path length. By incorporating greater mass onto a biosensor surface, the
optical density of binding partners on the surface is also increased,
thus rendering a greater resonant wavelength shift than would occur
without the added mass. The addition of mass can be accomplished, for
example, enzymatically, through a "sandwich" assay, or by direct
application of mass to the biosensor surface in the form of appropriately
conjugated beads or polymers of various size and composition. This
principle has been exploited for other types of optical biosensors to
demonstrate sensitivity increases over 1500× beyond sensitivity
limits achieved without mass amplification. See, e.g., Jenison et al.,
"Interference-based detection of nucleic acid targets on optically coated
silicon," Nature Biotechnology 19: 62-65 (2001).

[0134]As an example, a NH2-activated biosensor surface can have a
specific capture element comprising a single-strand DNA capture probe
immobilized on the surface. The capture probe interacts selectively with
its complementary target binding partner. The binding partner, in turn,
can be designed to include a sequence or tag that will bind a "detector"
molecule. A detector molecule can contain, for example, a linker to
horseradish peroxidase (HRP) that, when exposed to the correct enzyme,
will selectively deposit additional material on the biosensor only where
the detector molecule is present. Such a procedure can add, for example,
300 angstroms of detectable biomaterial to the biosensor within a few
minutes.

[0135]A "sandwich" approach can also be used to enhance detection
sensitivity. In this approach, a large molecular weight molecule can be
used to amplify the presence of a low molecular weight molecule. For
example, a binding partner with a molecular weight of, for example, about
0.1 kDa to about 20 kDa, can be tagged with, for example,
succinimidyl-6-[a-methyl-a-(2-pyridyl-dithio) toluamido] hexanoate
(SMPT), or dimethylpimelimidate (DMP), histidine, or a biotin molecule.
Where the tag is biotin, the biotin molecule will binds strongly with
streptavidin, which has a molecular weight of 60 kDa. Because the
biotin/streptavidin interaction is highly specific, the streptavidin
amplifies the signal that would be produced only by the small binding
partner by a factor of 60.

[0136]Detection sensitivity can be further enhanced through the use of
chemically derivatised small particles. "Nanoparticles" made of colloidal
gold, various plastics, or glass with diameters of about 3-300 nm can be
coated with molecular species that will enable them to covalently bind
selectively to a binding partner. For example, nanoparticles that are
covalently coated with streptavidin can be used to enhance the visibility
of biotin-tagged binding partners on the biosensor surface. While a
streptavidin molecule itself has a molecular weight of 60 kDa, the
derivatised bead can have a molecular weight of any size, including, for
example, 60 KDa. Binding of a large bead will result in a large change in
the optical density upon the biosensor surface, and an easily measurable
signal. This method can result in an approximately 1000×
enhancement in sensitivity resolution.

[0137]A feature of one of the possible biosensor platforms used in the
methods of this disclosure is that light energy entering the optically
transparent layer is diffracted out of the layer immediately due to the
nature of the corrugated platform. Therefore no or negligible waveguiding
occurs. Typically the propagation distance is 100 μm or less,
preferably 10 μm or less. This is a very surprisingly short distance.
The propagation distance is the distance over which the energy of the
radiation is reduced to 1/e.

[0138]The range of angles suitable for creating a resonance condition is
limited by the angle of total reflection for incident light on the
platform. Preferred angles are less than 45°, e.g. 30° or
less, e.g. 20° to 10° or below, e.g. 0.1° to
9.9°. The angle may equal or approximate normal incidence.

[0139]The light generating means may comprise a laser for emitting a
coherent laser beam. Other suitable light sources include discharge lamps
or low pressure lamps, e.g. Hg or Xe, where the emitted spectral lines
have sufficient coherence length, and light-emitting diodes (LED). The
apparatus may also include optical elements for directing the laser beam
so that it is incident on the platform at an angle θ, and elements
for shaping the plane of polarisation of the coherent beam, e.g. adapted
to transmit linearly polarised light. The angle θ may be defined by
the expression sin θ=n-λ/Λ where Λ is a period
of the diffractive grooves, λ is the wavelength of the incident
light and n is the effective refractive index of the optically
transparent layer.

[0140]Examples of lasers that may be used are gas lasers, solid state
lasers, dye lasers, semiconductor lasers. If necessary, the emission
wavelength can be doubled by means of nonlinear optical elements.
Especially suitable lasers are argon ion lasers, krypton ion lasers,
argon/krypton ion lasers, and helium/neon lasers which emit at
wavelengths between 275 and 753 nm. Very suitable are diode lasers or
frequency doubled diode lasers of semiconductor material which have small
dimensions and low power consumption.

[0141]Another appropriate type of excitation makes use of VCSEL's
(vertical cavity surface emitting lasers) which may individually excite
the recognition elements on the platform.

[0142]In one embodiment, a support to be used in a method of the invention
will be illuminated with white light that will contain light of every
polarisation angle. The orientation of the polarisation angle with
respect to repeating features in a biosensor grating will determine the
resonance wavelength. For example, a "linear grating" biosensor structure
consisting of a set of repeating lines and spaces will have two optical
polarisations that can generate separate resonant reflections. Light that
is polarized perpendicularly to the lines is called "s-polarized," while
light that is polarized parallel to the lines is called "p-polarized."
Both the s and p components of incident light exist simultaneously in an
unfiltered illumination beam, and each generates a separate resonant
signal. A support structure can generally be designed to optimize the
properties of only one polarisation (the s-polarisation), and the
non-optimized polarisation is easily removed by a polarizing filter.

[0143]In order to remove the polarisation dependence, so that every
polarisation angle generates the same resonant reflection spectra, an
alternate structure can be used that consists of a set of concentric
rings. In this structure, the difference between the inside diameter and
the outside diameter of each concentric ring is equal to about one-half
of a grating period. Each successive ring has an inside diameter that is
about one grating period greater than the inside diameter of the previous
ring. The concentric ring pattern extends to cover a single sensor
location--such as a microarray spot or a microtitre plate well. Each
separate microarray spot or microtitre plate well has a separate
concentric ring pattern centred within it. All polarisation directions of
such a structure have the same cross-sectional profile. The concentric
ring structure must be illuminated precisely on-centre to preserve
polarisation independence. The grating period of a concentric ring
structure is less than the wavelength of the resonantly reflected light.
The grating period is about 0.01 micron to about 1 micron. The grating
depth is about 0.01 to about 1 micron.

[0144]In another embodiment, an array of holes or posts are arranged to
closely approximate the concentric circle structure described above
without requiring the illumination beam to be centred upon any particular
location of the grid. Such an array pattern is automatically generated by
the optical interference of three laser beams incident on a surface from
three directions at equal angles. In this pattern, the holes (or posts)
are centred upon the corners of an array of closely packed hexagons. The
holes or posts also occur in the centre of each hexagon. Such a hexagonal
grid of holes or posts has three polarisation directions that "see" the
same cross-sectional profile. The hexagonal grid structure, therefore,
provides equivalent resonant reflection spectra using light of any
polarisation angle. Thus, no polarizing filter is required to remove
unwanted reflected signal components. The period of the holes or posts
can be about 0.01 μm to about 1 μm and the depth or height can be
about 0.01 μm to about 1 μm.

[0145]The detecting system (see FIG. 7 below for one example) may be
arranged to detect luminescence such as fluorescence. Affinity partners
can be labelled in such a way that Forster fluorescence energy transfer
(FRET) can occur upon binding of analyte molecules to capture molecules.
The maximum of the luminescence intensity might be slightly shifted
relative to the position of highest anormal reflection depending on the
refractive index values of the layer system and the corresponding Fresnel
Coefficients.

[0148]The sample may also comprise constituents that are not soluble in
the solvents used, such as pigment particles, dispersants and natural and
synthetic oligomers or polymers.

[0149]Luminescent labels can be used to modify capture elements, assayed
molecules in the analyte, or any other species, e.g.
endogeneous/exogeneous controls, spacer molecules, primers,
bio/materials, which interact with the sensor surface.

[0150]The luminescence dyes used as markers may be chemically or
physically, for instance electrostatically, bonded to one or multiple
affinity binding partners (or derivatives thereof) present in the analyte
solution and/or attached to the platform. In case of naturally-occurring
oligomers or polymers such as DNA, RNA, saccharides, proteins, or
peptides, as well as synthetic oligomers or polymers, involved in the
affinity reaction, intercalating dyes are also suitable. Luminophores may
be attached to affinity partners present in the analyte solution via
biological interaction such as biotin/avidin binding or metal complex
formation such as His-tag coupling.

[0151]One or multiple luminescence markers may be attached to affinity
partners present in the analyte solution, to capture elements immobilized
on the platform, or both to affinity partners present in analyte solution
and capture elements immobilized at the platform, in order to
quantitatively determine the presence of one or multiple affinity binding
partners.

[0152]The spectroscopic properties of the luminescence markers may be
chosen to match the conditions Forster Energy Transfer or Photoinduced
Electron Transfer. Distance and concentration dependent luminescence of
acceptors and donors may then be used for the quantification of analyte
molecules.

[0153]Quantification of affinity binding partners may be based on
intermolecular and/or intramolecular interaction between such donors and
acceptors bound to molecules involved in affinity reactions.
Intramolecular assemblies of luminescence donors and acceptors covalently
linked to affinity binding partners, Molecular Beacons (S. Tyagi et al.,
Nature Biotechnology) which change the distance between donor and
acceptor upon affinity reaction, may also be used as capture molecules or
additives for the analyte solution. In addition, pH and potentially
sensitive luminophores or luminophores sensitive to enzyme activity may
be used, such as enzyme mediated formation of fluorescing derivatives.

[0154]Transfluorospheres or derivatives thereof may be used for
fluorescence labelling, and chemiluminescent or electroluminescent
molecules may be used as markers.

[0155]Luminescent compounds having luminescence in the range of from 400
nm to 1200 nm which are functionalised or modified in order to be
attached to one or more of the affinity partners, such as derivatives of:
[0156]polyphenyl and heteroaromatic compounds [0157]stilbenes,
[0158]coumarines, [0159]xanthene dyes, [0160]methine dyes, [0161]oxazine
dyes, [0162]rhodamines, [0163]fluoresceins, [0164]coumarins, stilbenes,
[0165]pyrenes, perylenes, [0166]cyanines, oxacyanines, phthalocyanines,
porphyrins, naphthalopcyanins, azobenzene derivatives, distyryl
biphenyls, [0167]transition metal complexes e.g. polypyridyl/ruthenium
complexes, tris (2,2' bipyridyl) ruthenium chloride,
tris(1,10-phenanthroline) ruthenium chloride, tris (4,7
diphenyl-1,10-phenanthroline) ruthenium chloride and
polypyridyl/phenazine/ruthenium complexes, such as
octaethyl-platinum-porphyrin, Europium and Terbium complexes may be used
as luminescence markers [0168]nanoparticles, microparticles [0169]any
other light emitting species that can be excited by evanescent fields.

[0170]Suitable for analysis of blood or serum are dyes having absorption
and emission wavelength in the range from 400 nm to 1000 nm. Furthermore
luminophores suitable for two and three photon excitation can be used.

[0171]Dyes which are suitable in this invention may contain functional
groups for covalent bonding, e.g. fluorescein derivatives such as
fluorescein isothiocyanate or NH2 esters. Also suitable are the
functional fluorescent dyes commercially available from Amersham Life
Science, Inc., Texas, and Molecular Probes Inc. Other suitable dyes
include dyes modified with deoxynucleotide triphosphate (dNTP) which can
be enzymatically incorporated into RNA or DNA strands.

[0172]Further suitable dyes include Quantum Dot Particles or Beads
(Invitrogen Corporation, Carlsbad Calif.) or derivatives thereof or
derivatives of transition metal complexes which may be excited at one and
the same defined wavelength, and derivatives show luminescence emission
at distinguishable wavelengths.

[0173]Analytes may be detected either via directly bonded luminescence
markers, or indirectly by competition with added luminescence marked
species, or by concentration-, distance-, pH-, potential- or redox
potential-dependent interaction of luminescence donors and
luminescence/electron acceptors used as markers bonded to one and/or
multiple analyte species and/or capture elements. The luminescence of the
donor and/or the luminescence of the quencher can be measured for the
quantification of the analytes.

[0174]In the same manner affinity partners can be labelled in such a way
that electron transfer or photoinduced electron transfer leads to
quenching of fluorescence upon binding of analyte molecules to capture
molecules.

[0175]Appropriate detectors for luminescence include CCD-cameras,
photomultiplier tubes, avalanche photodiodes, photodiodes, hybrid
photomultiplier tubes, or arrays thereof. Detection can also be performed
in the absence of a label as described in U.S. patent application
publications U.S. 2003/0027327; 2002/0127565, 2003/0059855 and
2003/0032039, or the U.S. Pat. No. 7,023,544, which are hereby
incorporated herein by reference. The detection means can be arranged to
detect in addition changes in refractive index. The incident beam may be
arranged to illuminate the sensing area or all sensing areas on one
common platform. Alternatively the beam can be arranged to illuminate
only a small sub-area of the sensing area to be analysed and the beam
and/or the platform may be arranged so that they can undergo relative
movement in order to scan the sensing area of the platform.

[0176]Accordingly, the detecting means may be arranged in an appropriate
way to acquire the luminescence signal intensities of the entire sensing
area in a single exposure step. Alternatively the detection and/or
excitation means may be arranged in order to scan the sensing areas
stepwise.

[0196]The activity or density of the capture molecules can be optimised in
a number of ways well known in the art.

[0197]Periodic structures can also be obtained by attaching nanoparticles
to a surface, wherein said nanoparticles are of similar size and act as a
periodical structure that allow optical coupling/resonance.

[0198]Several detection systems 300 for obtaining both BIND and ER data
from a grating-based biosensor are described in published PCT application
WO 2007/019024. One of them is illustrated in FIG. 7. The system 300 of
FIG. 7 is an imaging readout system. The biosensor 100 is designed to
exhibit both a sharp resonant peak, in the optical spectrum, for
label-free detection and a high electromagnetic field in the evanescent
region of the biosensor for significant enhancement of fluorescence
signal. The readout system reads out both of these effects, taking
advantage of these biosensor properties. This readout system has the
capability to measure either or both signals from the biosensor.

[0199]The ER+BIND biosensor 100, referred to herein as a "comBIND sensor"
herein, is interrogated optically from the bottom side of the sensor. On
the topside of the biosensor 100, the biosensor may be immersed in water
or another liquid, or it may be exposed to air. Any molecular or cellular
binding interaction, which the biosensor is designed to detect, takes
place on the topside of the biosensor 100. The biosensor 100 may be part
of a larger assay device that includes liquid containing vessels, such as
for example a microwell plate having e.g., 8 columns of wells, each row
containing 12 wells. The biosensor may also be a component of a
microarray slide (see FIG. 8). In the illustration of FIG. 7, a single
well (detection region) 302 is shown in cross-section, it being
understood that dozens, hundreds or even thousands of such detection
regions may be present.

[0200]The imaging readout and detection system 300 includes an ER light
source 340 in the form of a laser (e.g., HeNe laser), a broader spectrum
BIND light source 350 including as a halogen white light source or a LED
352, and a CCD camera system 338 serving as a common detector to capture
both ER and label-free data in successive images. The system 300 includes
an optical beam combining subsystem that includes dichroic mirrors 364
and 330 which serves to combine and direct incident light 372 from the
light sources 340 and 352 onto the biosensor. The dichroic mirror 330
collects signal light for detection and directs it to a lens 336 where it
is imaged by the CCD camera 338.

[0202]Signal detected by the CCD camera 338 through a lens system 336 is
processed electronically or by computer algorithm to become BIND
(label-free) data 380 or ER data 382. Such data may be stored, displayed,
and analyzed on an analytical instrument such as a computer or
workstation for the instrumentation shown in FIG. 7 (not shown, but
having access to data 382 and 380) by the user of the readout system 300.
Furthermore, the combination of the BIND data 380 and the ER data 382
allows the user to gain information on binding interactions or cell
interactions that is unique to the novel biosensor 100.

[0203]In the illustrated design, the optical components 340, 350 and 330
are designed to produce a single beam 372 of incident radiation and the
biosensor is moved in X and Y directions to thereby sequentially obtain
data from all the wells 302 or binding sites on the biosensor 100
surface. Such motion may be produced by placing the biosensor 100 on an
X-Y motion stage (not shown), of which persons skilled in the art are
familiar. When a given well or binding site 302 is in position such that
the well 302 is in registry with the beam 372, in one embodiment the
light sources 340 and 350 are operated in succession (or selectively
allowed to direct radiation onto the biosensor) and first and second
images are captured by the CCD camera 338, one an ER image and the other
a BIND image. The successive collection of CCD images could be
facilitated by use of the beam selection mechanism 360 (such as a
shutter), which selectively allows light from either the source 340 or
the source 350 to pass to the dichroic mirror 330 and be reflected onto
the biosensor. Beam selection can also be done electronically, such as by
electronically controlling the on and off times of the light sources 340
and 350. Alternatively, both light sources could be activated at the same
time and the selection mechanism 360 operated to pass both beams so that
the incident beam 372 contains light from both sources. In this
situation, the CCD camera 338 would capture a single image containing
both ER and BIND information. Image processing techniques would then be
applied to the resultant image from the CCD camera 338 to extract the
BIND and ER components of the composite image. The shift in peak
resonance wavelength measured by the detection system is determined on a
pixel-by-pixel basis and the magnitude of such shifts is converted either
to colors or to relative brightness, or both, for purposes of rendering
the label-free image such as shown in FIG. 9B.

[0204]The ER light source 340 may be a laser, such as a helium-neon (HeNe)
laser. The laser beam 341 further goes through a beam-conditioning device
342 such as a beam expander. The beam expander 342 expands a small
diameter laser beam into a large diameter laser beam. The output beam 343
is collimated and linearly polarized. The biosensor produces the ER
effect in response to incident light at a specific polarization.
Polarization may be achieved by using a laser designed for producing a
linearly polarized output laser beam.

[0205]The BIND (label-free) light source 350 may consist of a halogen or
LED light source 352, and a monochromator 354 with a wavelength
adjustment mechanism 356. The light beam 353 emitted by the light source
352 is broadband in nature, while the light beam 355 at the exit port of
the monochromator 354 is monochromatic.

[0206]The output light beam 355 from the monochromator 354 is conditioned
by a beam conditioning device 358, which may be a collimator. A mirror
365 directs the light beam 349 from the output of the conditioning device
358 to the dichroic mirror 364. The combined light from the light sources
340 and 350 is shown at 366 where it is directed to the beam splitting
and combining assembly 330 which then directs it to the bottom surface of
the biosensor 100.

[0207]The BIND light source 350 may also consist of a tuneable laser. In
that case, the beam-conditioning device 358 is a beam expander. Note also
that a tuneable laser or flash lamp could serve as a single illumination
source for both BIND and ER measurements.

[0208]In addition, since polarized light facilitates detection of a BIND
signal, there may be a polarizer within the light source 352 so that the
light 363 is linearly polarized. Alternatively, the light-directing
element 365 may be a polarizing beam splitter to transform a randomly
polarized light 359 into a linearly polarized light 363.

[0209]For detection of the laser excited fluorescence signal, the beam
splitting and combining assembly 330 incorporates a set of optical
filters 332 and 334. Filter 332 is a dichroic filter that reflects the
laser light while transmitting fluoresced light from the sample. Filter
332 also functions as a beamsplitter in the BIND wavelength range, which
is 830 nm to 900 nm in one preferred design. Filter 334 only allows
transmission of light within two wavelength ranges: laser excited
fluorescence and the BIND wavelength range. An imaging lens 336 may be
used to collect the fluorescence light at the biosensor surface and focus
it on the focal plane of the CCD camera 338.

[0210]The design of FIG. 7 also includes rotation apparatus to rotate the
biosensor relative to the incident beam 372 for purposes of ER detection.
In one possible embodiment, a rotation device 331 is attached to the beam
splitting and combining assembly 330 and rotates the assembly 330 as
indicated by the arrows (thereby providing for rotation of the incident
beam about angle θ). In an alternative embodiment, rotation device
331 is omitted and instead a rotational device 333 is attached to the XY
motion stage which operates to rotate the XY motion stage (and biosensor
100 mounted thereon) relative to the (fixed) incident beam 372, as
indicated by the arrows to the left of device 333 in FIG. 26.

[0211]Additional lenses, mirrors and optical filters may be incorporated
into the readout system to achieve desired performance. Properly designed
optical filters may be used to eliminate undesired cross-talk between
BIND detection and ER detection. In addition, a beam selection mechanism
in the form of electronic or mechanical shutters 360 may be used to
properly synchronize light illumination and detection of the two
channels, so that only one light source illuminates the biosensor at a
given time, to eliminate any cross-talk.

[0212]A significant advantage of the biosensor readout system described in
FIG. 7 is that both BIND and ER data may be collectedly simultaneously
(or in rapid succession) at the same biosensor location. High-resolution
imaging methods are useful for high content bioassays such as cell-based
assays or microarrays.

[0213]An integrating single point detector may replace the CCD camera 338.
In that case, the system produces an image by synchronizing sensor
motion, over the location of the incident radiation 372, with the
detector output.

[0214]Further details on use of a CCD camera to obtain ER data from a
biosensor can be found in the technical literature, e.g., an article of
Dieter Neuschafer, Wolfgang Budach, et al., Biosensors & Bioelectronics,
Vol. 18 (2003) p. 489-497, the contents of which are incorporated by
reference herein.

[0215]The following description and examples of calibration and
normalization methods for biosensors are merely illustrative of the
present disclosure and are not limiting.

EXAMPLES

[0216]Details on the ER slides ("NovaChip") have been published in e.g. D.
Neuschafer et al., Biosensors and Bioelectronics, 18: 489-497 (2003), or
W. Budach et al., Analytical Chemistry, 75: 2571-2577 (2003). The
approach takes advantage of a phenomenon that has been attributed as
abnormal reflection, see e.g. S. S. Wang & R. Magnusson, Applied Optics,
32(14): 2606-2613 (1993), or O. Parriaux et al., Pure & Applied Optics,
5: 453-469 (1996). Excitation photons incident on the chip under
resonance conditions couple into a thin corrugated metal oxide surface at
the site of incidence. As a result of the transducer geometry, the energy
is locally confined into the thin corrugated layer of high refractive
index material. Consequently, strong electromagnetic fields are generated
at the surface of the chip. The effect has been attributed as evanescent
resonance and leads to increased fluorescence intensity of chromophores
close to the surface. The effective field strength can be increased up to
100-fold by the confinement of the available excitation energy, depending
on the optical properties of the used optical detection system. The
NovaChips used for the experiments described in the examples have a
resonance angle of 2° with respect to normal for TE polarized
light of 633 nm wavelength. The observed increase of fluorescence
signal/noise ratio with the used Tecan microarray laser scanner was about
10-20 fold compared to glass slides processed under identical conditions.

[0217]FIG. 8 is a photograph of a biosensor captured by the CCD camera of
FIG. 7, the biosensor having the grating structure of FIG. 11 in the form
of a microarray having a multiplicity of capture element locations, the
locations clustered in groups of regions as indicated in FIG. 3.

[0218]FIGS. 9A-9C shows three different images of the spot or capture
element regions of the biosensor of FIG. 8, in which individual capture
element locations are visualized with a circle in FIGS. 9A, 9B, and 9C.

[0219]FIG. 9A is a "salt image" of one of the regions of FIG. 8 which
shows the printed spots or locations that are expected to consist of
capture elements (e.g. oligonucleotide) and print buffer. The actual
morphology of the spot locations in the image is varying and depends on
the process conditions. The image provides only qualitative information,
e.g. spots where capture materials have not been printed/deposited
correctly in the chip surface. This information can be used as basic
quality flags for each of the capture elements.

[0220]FIG. 9B is a label-free image of the region of FIG. 9A, obtained
after a washing step that removes the excess material from the biosensor
surface. The intensity of the individual locations corresponds to the
amount of immobilized material of the oligonucleotides which are
immobilized/deposited in the individual locations on the microarray. The
intensity measurements ("BIND" data) are made using the system of either
FIG. 3 or 7, or possibly a different system including one with a
spectrometer as disclosed in PCT application WO 2007/019024.

[0221]FIG. 9C is an image obtained with the system of FIG. 7 in an ER mode
showing the intensity of the regions corresponds to the abundance of a
particular mRNA in the hybridised sample.

[0229]Subsequently, the microarrays were scanned with a Tecan laser
scanner (gain 120) using a 633 nm laser (red) as a light source. The
optical filter of the detection unit was removed to allow collection of
scattered light images, i.e. images of laser light directly scattered
from the physical profile of the spots composed in large part of salt
from the spotting buffer. These images are therefore also termed "Salt"
images. FIG. 9A shows a close-up of the microarray used for this
application example. The images allow a coarse first assessment of the
microarray quality, as analysis of this image allows identification of
missing spots, particles or artifacts.

[0230]2. Label-Free Mode, Quantification

[0231]The microarray was then washed to remove unbound oligonucleotides
and spotting buffer salt from. Bound oligonucleotides remain attached to
reactive groups on the surface. The wash process consisted of 20 seconds
in a stirrer-containing recipient with Wash Buffer followed by 20 seconds
in a second recipient containing Wash Buffer diluted 1:3 with deionised
water. Subsequently, the slides were dried in a nitrogen stream.

[0232]After washing, a 16 Bit TIF label free image of the remaining
oligonucleotide material was obtained using a BIND TM Scanner from SRU
Biosystems Inc. The label free scan images the spectral shift of the TM
resonance at 15 um/pixel resolution. FIG. 9B shows the area of the label
free image that corresponds to the "salt" image of FIG. 9A.

[0233]Label Free Scanner Principle of Operation

[0234]The label free scanner system includes a light source and optical
elements that direct collimated white light towards the surface of the
sensor. An imaging spectrometer receives light reflected from the sensor
and generates an image where one axis represents a spatial line scan and
the second axis reports the reflected spectrum at each pixel on the line
scan. Software determines the spectral location of the TM resonance,
referred to as Peak Wavelength Value (PWV), within the reflected
spectrum. The line scan traversed the slide progressively constructing a
PWV image. Pixels corresponding to oligonucleotide spots have higher PWV
than areas without bound material. The label free signal consists of the
PWV difference (shift) between pixels corresponding to spots and
surrounding (background) pixels. Alternatively or in addition to this
analysis, one can acquire a baseline PWV scan prior to spotting and
subtract the baseline PWV values from a post spotted image. The shifts in
PWV are converted to color or intensity for purposes of rendering an
image such as shown in FIG. 9B.

[0235]Sample/RNA Processing

[0236]Commercial human reference RNA (Stratagene) was labelled per the
protocol and materials of the Ambion labelling kit (#1753). This
experiment used 100 ng of labelled RNA.

[0238]A Tecan HS 4800 Hybridisation station from Tecan, Inc. performed the
prewash, hybridisation, and postwash of the microarrays. Prewash
consisted of 3 cycles with Wash Buffer at RT/75° C./50° C.
(each 20 sec) followed by an additional 10 sec wash with Hybridisation
Buffer at 50° C. A 100 μL sample of 100 ng concentration
labelled RNA in Hybridisation Buffer was then injected into the flow
chamber at 50° C. and agitated for 1 minute. After 10 min. at
75° C., the temperature was maintained at 42° C. for 16
hours with agitation. The postwash consisted of 4 cycles with Wash Buffer
at 42° C. followed by an additional wash at 23° C. (each 20
sec). Finally, the slides were washed 3 times with diluted Wash Buffer at
RT, dried in N2 stream, and scanned immediately with a Tecan
fluorescence laser scanner (gain 80%). Images were stored as 16 Bit TIF
files. FIG. 9c shows the reference area of the fluorescence mode image
that corresponds to the previously discussed "salt" (FIG. 9A) and
label-free (FIG. 9B) images.

[0239]Image Processing and Quality Control

[0240]The spots or capture element regions, are circumscribed graphically
by the analysis software (ArrayPro) in FIGS. 9A, 9B and 9C.

[0241]In FIG. 9A, the scatter mode, "salt image", images printed spots
expected to result in oligonucleotide capture elements after washing.
Actual spot morphology varies depending on process conditions. The image
provides only qualitative information regarding the presence and quality
of an individual capture element, e.g. whether the printing process
deposited material at the specified location. This information can serve
as a flag for gross printing errors.

[0242]In FIG. 9B, the label-free mode image, is obtained after a washing
step that removes excess (unbound) material from the spotted areas. The
intensity of the label free signal in the individual capture areas
corresponds to the amount of immobilized material (oligonucleotide).
Thus, this signal provides a quantitative value for the amount of probe
material available for binding in the subsequent hybridisation step.

[0243]In FIG. 9C, the ER/fluorescence mode image, the signal intensity of
each capture area (spot) corresponds to the abundance of labelled mRNA
bound to the probe material in that area.

[0244]The images obtained in label-free and luminescence/fluorescence mode
were analyzed by means of Array Pro (Mediacybernetics, Inc. US). A sub
section of 10 Rows (R)×10 Columns (C) was used for the present
example (same section for all images to allow comparison and
calibration). The three TABLES 1a, 1b, and 1c below quantify data from
Row 1 of the 10×10 spot array depicted in images FIGS. 9A, 9B and
9C. The TABLES derive "Net" intensity by subtracting the mean of a local
"Background" ring surrounding each spot from the interior mean ("Raw"
signal) of each spot.

[0245]TABLE 1a quantifies data obtained in by analysing the intensity of
the first 10 spots/genes (R=1, C=1 to 10) in the scatter or "salt" image.
Definition of an intensity threshold (or more sophisticated rules,
algorithms, in general) allows identification of incorrectly deposited
spots. See also in Table 2, where the scatter mode intensity threshold is
used into flag those spots/capture elements that appear missing. This
procedure can be automated and allows a first assessment of the quality
of a microarray production batch.

[0246]TABLE 1b reports data obtained in label-free mode for the first 10
spots/genes (R=1, C=1 to 10). Again, the definition of a threshold (or
more sophisticated rules, algorithms, in general) allows identification
of incorrectly printed spots. In addition, the label free Net intensity
provides a calibration of the deposited material.

[0247]For example, the first Oligonucleotide I (Row=1, Column=1),
Oligonucleotide (1,1), has a Net Signal of 4116 counts. Oligonucleotide
(1,7) has an intensity of 7359 counts, which indicates that more material
has been immobilized. These values, yielding relative probe density, at
each spot, provide the basis for normalizing subsequent fluorescent mode
signal. Oligonucleotide (1,5) has a very low Net signal of 245 counts,
which can be interpreted as a missing spot, i.e. the location does not
contain enough probe material to provide reliable data.

[0248]Automation of this procedure provides a rapid and detailed
assessment of the quality of a microarray print batch as well as
quantification of the density of printed genes immobilized within
individual capture elements.

[0249]TABLE 1c shows the corresponding fluorescence data for the same set
of oligonucleotides. The fluorescence signal responds to the frequency of
gene binding events during hybridisation. Both the density of printed
probe genes and the frequency of gene commonality between the probe and
analyte determine the frequency of gene binding and hence, both factors
affect fluorescent signal. For example, a low fluorescence signal could
result from a missing spot or negligible gene expression in the target
sample.

[0250]Applying a threshold to the Scatter net intensities column in the
table above flags exceptionally weak or missing spots. Net intensities
from the label free scan provide quantifiable information regarding the
amount of immobilized capture element at each position. For example,
oligonucleotides (1,5) and (3,1) have very low values. Again, the
definition of a threshold allows flagging of missing spots. Furthermore,
normalization of fluorescent signal by the label free intensities can
compensate for variations of the amount of immobilized material. The
third section of Table 2 accomplishes this normalization process.

[0251]Application of the following Normalization Equation normalizes the
fluorescence (also termed luminescence) signal (LNIcalibrated) to
the amount of probe material as determined by the label free scan:

LNIcalibrated=LNInc*Scaling Value/LFNI, Equation (1)

where LNInc represents the non-calibrated fluorescence signal and
LFNI represents the label free net intensity. A scaling or target label
free value of 5000 cts maintains the fluorescent signal count range.
Calibrated values reduce variability in the assessment of gene expression
level by decreasing/compensating for the influence of spot printing
variability. Though not shown in this example, a "salt" image may
indicate successful deposition of buffer solution but not result in the
immobilization of actual probe material, either through an error in
buffer composition or process artifact. The label free signal reports
actual probe immobilization level immediately prior (in this example) to
the labelled analyte binding event.

[0252]FIG. 10 illustrates alternative forms of a biosensor platform,
including: discs, concentric designs, etc., as shown in International
Patent Application WO 01/02839. The sensing elements (see FIG. 10a) can
be arranged in various ways, for instance rectangular, circular,
hexagonal-centric, ellipsoidal, linear or labyrinthine. The sensing area
(see FIG. 10b) may be rectangular, round or of any other shape. The
grooves may be arranged either equidistant linear or equidistant
circular, or may correspond to segments of such structures. The platform
(see FIGS. 10c to 10f) can be either rectangular or disc-shaped, or of
any other geometry. The platform can comprise one or multiple sensing
areas, each sensing area can comprise one or multiple capture elements,
and each capture element can comprise one or multiple labeled or
unlabelled capture molecules. The platform can also be adapted to
microtitre-type plates/devices (see FIG. 10g) in order to perform one or
multiple assays in the individual microtitre wells. This can be achieved
for all plate types: 96, 384, 1536, or higher numbers of wells,
independently of the dimensions of the respective microtitre-plate.

[0253]FIG. 11 shows a schematic view of an ER sensor similar to that shown
in FIGS. 8 and 9.

[0254]FIG. 12 shows a schematic view of an ER/BIND composite sensor.

[0255]FIGS. 13A and B are two views of a unit cell of an ER/BIND composite
sensor as shown in FIG. 12, where the grating depth<thickness of high
refractive index layer.

[0256]FIGS. 14A and 14B are two views of a unit cell of an ER/BIND
composite sensor as shown in FIG. 12, where the grating
depth>thickness of high refractive index layer.

[0257]From the above discussion, it will be appreciated that we have
disclosed a method for assessing the immobilization quality and/or
quantity of probes or an array of probes immobilized on a biosensor
having a periodic grating structure and a multitude of probe locations on
a surface thereof, wherein the immobilization quality and/or quantity of
the immobilized probes is assessed individually at each probe location in
a spatially resolved manner prior to the binding of an analyte to the
probes,

[0258]said method comprising the steps of: [0259](1) obtaining
two-dimensional data and/or images from said biosensor by: [0260](A) in
an Evanescent Resonance mode, exciting of bound luminescence labels and
collecting data of the resulting emissions from said biosensor, and
[0261](B) in a label-free mode, obtaining a two dimensional image of the
biosensor surface and peak wavelength value (PWV) data for the portions
of the two-dimensional image which comprise images of probe locations of
the biosensor, the peak wavelength value comprising the peak wavelength
of light reflected from the biosensor due to resonant coupling of light
into the biosensor; and [0262](2) characterizing the immobilization
quality and/or quantity of the probes or array of probes immobilized on
the biosensor surface from the two-dimensional data and/or images.

[0263]In one embodiment, the biosensor comprises a multitude of sample
regions (FIGS. 8, 9A-C), each sample region potentially containing
biological material bound to the biosensor, and wherein the array is
formed as a surface of a periodic grating structure (FIG. 1-6), and
wherein step (1) of the method comprises the steps of: [0264](1)
obtaining a two-dimensional image of the biosensor (FIG. 8); [0265](2)
obtaining peak wavelength value (PWV) data for the portions of the
two-dimensional image which comprise images of sample regions of the
biosensor (FIG. 9B), the peak wavelength value comprising the peak
wavelength of light reflected from the biosensor due to resonant coupling
of light into the grating structure; and [0266](3) obtaining quantitative
information as to the amount of binding of the biological material to the
sample regions of the array from the peak wavelength value data (e.g.,
obtained from BIND data, see also Tables 1B, 2).

[0267]The probes may be deposited on the biosensor using any convenient
and known method, including use of a printer, piezo-array printer or pin
printer.

[0268]Further, the biosensor may be attached to an internal surface of a
liquid containing vessel, such as a bottomless microwell plate, a test
tube, a Petri dish and a microfluidic channel.

[0269]In another aspect, a method for assessing the immobilization quality
and/or quantity of probes or an array of probes immobilized on a
biosensor having a periodic grating structure and a multitude of probe
locations on a surface thereof, wherein the immobilization quality and/or
quantity of the probes is assessed individually at each probe location in
a spatially resolved manner prior to the binding of an analyte to the
probes, said method comprising the steps of: [0270](1) measuring peak
wavelength value (PWV) data of the probe locations of the biosensor;
[0271](2) obtaining a 2-dimensional image of the probe locations in a
spatially resolved manner (PWV images) (FIG. 9B), [0272](3) obtaining
quantitative information of the immobilization quality and/or quantity of
probes immobilized on the biosensor from the PWV data (e.g., obtained
from BIND data, see also Tables 1B, 2).

[0273]In the above method, the method may further comprise the steps of:
[0274]1) applying a labelled sample to the multitude of probe locations;
[0275]2) obtaining evanescent resonance (ER) measurements of the
multitude of probe locations (FIG. 9C); and [0276]3) normalizing the ER
measurements with the quantitative information obtained.In one embodiment
the normalization of the ER measurements is according to normalizing
equation (1).

[0277]In one embodiment, the PWV data and images are indicative for the
amount and morphology of potentially immobilized material on the surface
of the biosensor. The method further comprises the step of using the PWV
data and images to calibrate data and images obtained after hybridization
of a sample to the immobilized material.

[0278]In some embodiments of the method of this disclosure, the method may
further include the steps of acquiring label free PWV data and images
(FIG. 9B) and/or ER measurements (FIG. 9C) at one or more stages in a
process of manufacture of the biosensor, including one or more of the
following stages: biosensor surface cleaning, biosensor surface
modification, immobilization of materials onto the biosensor surface,
biosensor wash steps, biosensor drying steps, and hybridization of a
sample on the surface of the biosensor.

[0279]Additionally, the method may further comprise the step of correcting
data and images obtained after hybridization (FIG. 9C) based on the
pre-hybridisation PWV images/data (FIG. 9B), thus
calibrating/compensating for variations of amount and morphology of
immobilized capture material immobilised on the biosensor.

[0280]In some embodiments, the biosensor takes the form of a substrate
(e.g., polyester or MYLAR sheet) and wherein the surface of the biosensor
is coated with a layer of high index of refraction material (e.g.,
TiO2) consisting of material having a refractive index n2
higher than that of the substrate n1, wherein the depth of the layer
is between 10 and 1000 nm, and the resulting periodicity is in the range
of 100 to 1000 nm, and the substrate is of planar, cylindrical, conical,
spherical, or elliptical geometry.

[0281]In some embodiments, a salt image (FIG. 9A) is further obtained and
analysed. The salt image resulting from the method of spotting the probes
to be immobilised to the support, said method comprising the steps of
spotting a salt containing-solution containing the probes to be
immobilised to the support, optionally drying said salt
containing-solution containing the probes, and obtaining an image of the
locations where the probes should have been immobilised, said image being
obtained prior to any washing step, so that the absence of salt at a
specific location indicates that spotting of the probe to be immobilised
at this specific location has not occurred at said specific location.

[0283]The biosensor preferably includes an optically transparent layer
which is formed from inorganic material selected from the group
consisting of a metal oxide such as Ta2O5, TiO2,
Nb2O5, ZrO2, ZnO or HfO2; organic materials selected
from the group consisting of polyamide, polyimide, PP, PS, PMMA,
polyacryl acids, polyacryl esters, polythioether, or
poly(phenylenesulfide); and derivatives thereof. The optically
transparent layer can be either the periodic surface grating material
layer or the high index of refraction dielectric material deposited on
the grating.

[0284]In the methods of this disclosure, the immobilized probes or array
of probes are labelled with at least one of the following: spacers
molecules, energy-donors, energy-acceptors, electron-donors,
electron-acceptors, chromophores, luminophores, fluorophores,
phosphorescence labels, spectroscopic labels, biological functions, or
chemical modifications. In other embodiments, the array of probes are
unlabeled.

[0286]In some embodiments, the array of probes deposited on the biosensor
are unlabeled. In this case, evanescent resonance (ER) data/images (FIG.
9C) are obtained from the biosensor and calibrated using the quantitative
information obtained from BIND measurements or images obtained from the
sensor.

[0287]In the methods of this disclosure, the methods may further include a
step of obtaining a spectrum for background signals produced by the
biosensor and wherein the characterization of the immobilization quality
and/or quantity of probes is made after subtraction of the spectrum for
background signals produced by the biosensor.

[0288]In still another aspect, a method of detecting and/or quantifying an
analyte using an biosensor (FIG. 8) comprising an array of immobilised
probes on a surface thereof, the biosensor constructed in the form of a
periodic grating structure, wherein the presence and/or concentration of
said analyte is normalised with respect to the presence and/or
concentration of said immobilised probes as described above, wherein said
presence and/or concentration of said immobilised probes is assessed at
locations of the array prior to the potential binding of the analyte to
the surface of the biosensor. The presence and/or concentration of the
immobilized probes is assessed using a two dimensional image of the
biosensor surface and peak wavelength value (PWV) data for the portions
of the two-dimensional image which comprise images of probe locations of
the biosensor, the peak wavelength value comprising the peak wavelength
of light reflected from the biosensor due to resonant coupling of light
into the biosensor The method may further include the step of obtaining a
spectrum for background signals produced by the biosensor and wherein the
normalization is performed after subtraction of said spectrum for
background signals produced by the biosensor.

[0289]The method may also further include the steps of: [0290](1)
processing the biosensor to prepare for a hybridisation of a sample,
[0291](2) hybridizing the sample to the biosensor; and [0292](3)
recording a post-hybridisation image of the biosensor, where the
resulting image represents the bound sample.

[0293]This method may further include a step of recording a
post-hybridisation label free image (FIG. 9B) in a label-free mode of the
biosensor.

[0294]The method may further include a step of correcting the
post-hybridisation image based on the pre-hybridisation data obtained,
thus compensating for capture material variations on the biosensor.

[0296]In another aspect, a non-contact method of qualitative analysis of a
microarray chip, comprising the steps of: [0297](a) providing a
microarray chip (FIG. 8) in the form of a multitude of sample regions 802
on a surface of a periodic grating structure (FIGS. 1-7); [0298](b)
depositing of capture elements to the grating structure; [0299](c)
obtaining a two-dimensional image of the microarray chip (FIG. 8, 9A);
[0300](d) obtaining peak wavelength value (PWV) data (FIG. 9B) for the
portions of the two-dimensional image which comprise images of sample
regions of the microarray chip, the peak wavelength value comprising the
peak wavelength of light reflected from the microarray due to resonant
coupling of light into the grating structure; and [0301](e) obtaining
qualitative information as to the binding of the capture elements to the
sample regions of the microarray from either (1) the two-dimensional
image or (2) the peak wavelength value data.In this method, the capture
elements can be deposited using a piezo-array or pin printer.

[0302]The qualitative information obtained in step e) characterizing the
binding of the capture elements as a function of the position on the
surface of the biosensor. The capture elements can vary, and in one
embodiment are selected from the group of materials consisting of a
nucleic acid material and a protein.

[0303]In yet another aspect of this disclosure, a method of analysis of a
microarray chip (FIG. 8) is provided comprising the steps of: [0304](a)
providing a microarray chip in the form of a multitude of sample regions
802 on a surface of a periodic grating structure (FIG. 1-7); [0305](b)
applying a biological material to the sample regions; [0306](c) obtaining
a two-dimensional image of the microarray (FIG. 8, 9A); [0307](d)
obtaining peak wavelength value (PWV) data (FIG. 9B) for the portions of
the two-dimensional image which comprise images of sample regions of the
microarray, the peak wavelength value comprising the peak wavelength of
light reflected from the microarray due to resonant coupling of light
into the grating structure; [0308](e) performing a hybridisation step
comprising applying a second sample material to the sample regions;
[0309](f) obtaining a two-dimensional image of the microarray after the
hybridisation step (FIG. 9C); and [0310](g) obtaining peak wavelength
value (PWV) data for the portions of the two-dimensional image which
comprise images of sample regions of the microarray after the
hybridisation step.

[0311]In one embodiment of this method the hybridisation step comprises
the step of applying a fluorescent probe to the biological material.

[0312]The method may further include a step of obtaining evanescent
resonance (ER) measurements of the sample regions after the hybridisation
step. In particular, the method may include obtaining ER measurements
from the microarray chip and normalizing the measurements with reference
to quantitative data of the amount of biological material bound to the
sample regions obtained from the peak wavelength value (PWV) data
obtained in step d). The biological material adhered to a biosensor can
take the form of a DNA microarray.

[0313]In still another aspect, a method for the determination of the
amount of DNA adhered to a biosensor following a hybridisation protocol
is disclosed, in which the method comprises the combined use of
label-free and label methods, as described herein.

[0314]The following are definitions of terms which will be used in the
description: [0315]"Platform", "biosensor" or "support": a whole
transducer/chip containing one or a plurality of sensing areas.
[0316]"Peak wavelength value (PWV)": position and intensity of the
resonance generated with incident light incident on the periodical
structure under resonance conditions, as measured by a detection system.
In other words, measurement of the PWV comprises measurement of the
position and intensity of the transmitted beam or of the abnormal high
reflection at each location. [0317]"Sensing area": a whole corrugated
area capable of creating an evanescent field by a resonance effect and
containing one or a plurality of capture elements. [0318]"Capture
element", "location" or "probe": an individual sensing spot containing
one or a variety of species of capture molecules. [0319]The expressions
"location of the array" or "regions of the support" is not to be
understood as being limited to a whole spot of deposited/attached
material. The smallest size of said "regions of the support" depends on
the optical resolution of the used detection system. In other words, the
method of the invention can be carried out e.g. (scanning) pixel-wise.
[0320]"Orientation" is understood to mean that the electric field vector
of the linearly polarized light is parallel or perpendicular to the
periodic surface grating of the biosensor. [0321]"Coherent light" is
understood to mean that the coherence length of the radiation, i.e. the
spatial extent to which the incident beam has a defined phase relation,
is large compared to the thickness of the biosensor.

EQUIVALENTS

[0322]The present invention is not to be limited in terms of the
particular embodiments described in this application, which are intended
as single illustrations of individual aspects of the invention. Many
modifications and variations of this invention can be made without
departing from its spirit and scope, as will be apparent to those skilled
in the art. Functionally equivalent methods and apparatuses within the
scope of the invention, in addition to those enumerated herein, will be
apparent to those skilled in the art from the foregoing descriptions.
Such modifications and variations are intended to fall within the scope
of the appended claims. The present invention is to be limited only by
the terms of the appended claims along with the full scope of equivalents
to which such claims are entitled.